U.S. patent application number 13/989738 was filed with the patent office on 2014-02-06 for diagnostic and/or screening agents and uses therefor.
This patent application is currently assigned to IMMUNEXPRESS PTY LTD. The applicant listed for this patent is Richard Bruce Brandon, Glenn Stone, Mervyn Rees Thomas. Invention is credited to Richard Bruce Brandon, Glenn Stone, Mervyn Rees Thomas.
Application Number | 20140037649 13/989738 |
Document ID | / |
Family ID | 46145307 |
Filed Date | 2014-02-06 |
United States Patent
Application |
20140037649 |
Kind Code |
A1 |
Brandon; Richard Bruce ; et
al. |
February 6, 2014 |
DIAGNOSTIC AND/OR SCREENING AGENTS AND USES THEREFOR
Abstract
Disclosed are methods and apparatus for diagnosis, detection of
host response, monitoring, treatment or management of sepsis,
infection-negative systemic inflammatory response syndrome (SIRS)
and post-surgical inflammation in mammals. More particularly, the
present invention discloses marker genes and their splice variant
transcripts as well as their expression products, which are useful
for distinguishing between sepsis and infection-negative SIRS,
including post-surgical inflammation, and to the use of these
markers in grading, monitoring, treatment and management of these
conditions.
Inventors: |
Brandon; Richard Bruce;
(Boonah, AU) ; Thomas; Mervyn Rees; (Chapel Hill,
AU) ; Stone; Glenn; (Glenwood, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Brandon; Richard Bruce
Thomas; Mervyn Rees
Stone; Glenn |
Boonah
Chapel Hill
Glenwood |
|
AU
AU
AU |
|
|
Assignee: |
IMMUNEXPRESS PTY LTD
TOOWONG
AU
|
Family ID: |
46145307 |
Appl. No.: |
13/989738 |
Filed: |
November 24, 2011 |
PCT Filed: |
November 24, 2011 |
PCT NO: |
PCT/AU11/01540 |
371 Date: |
October 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61417381 |
Nov 26, 2010 |
|
|
|
Current U.S.
Class: |
424/164.1 ;
435/6.12; 506/9; 514/1.4 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 1/6883 20130101; C12Q 1/6876 20130101 |
Class at
Publication: |
424/164.1 ;
506/9; 435/6.12; 514/1.4 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for assessing whether a subject has, or is at risk of
developing, one of a plurality of conditions selected from sepsis,
infection-negative SIRS ("inSIRS") and post-surgical inflammation,
the method comprising: comparing the level of at least one
inflammatory response continuum (IRC) marker expression product of
a multi-transcript-producing gene in the subject to the level of a
corresponding IRC marker expression product in at least one control
subject selected from: a post-surgical inflammation-positive
subject, an inSIRS positive subject, a sepsis-positive subject and
a normal subject, wherein a difference between the level of the at
least one IRC marker expression product and the level of the
corresponding IRC marker expression product indicates whether the
subject has, or is at risk of developing, one of the conditions,
wherein the at least one IRC marker expression product is
predetermined as being differentially expressed between at least
two of the conditions and wherein at least one other expression
product from the multi-transcript producing gene is predetermined
as being not so differentially expressed.
2. A method according to claim 1, wherein the
multi-transcript-producing gene is selected from the group
consisting of: ankyrin repeat and death domain containing 1A
(ANKDD1A) gene, rho 2 (GABRR2) gene, orthodenticle homeobox 1
(OTX1) gene, pannexin 2 (PANX2) gene, rhomboid 5 homolog 2
(Drosophila) (RHBDF2) gene, SLAM family member 7 (SLAMF7) gene,
autophagy/beclin-1 regulator 1 (AMBRA1) gene, carboxylesterase 2
(intestine, liver) (CES2) gene, caseinolytic peptidase B homolog
(E. coli) (CLPB) gene, homeodomain interacting protein kinase 2
(HIPK2) gene and chromosome 1 open reading frame 91 (C1ORF91) gene,
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 (NDST1)
gene, solute carrier family 36 (proton/amino acid symporter)
(member 1 (SLC36A1) gene, ADAM metallopeptidase domain 19 (meltrin
beta) (ADAM19) gene, cullin 7 (CUL7) gene, thyroglobulin (TG) gene,
programmed cell death 1 ligand 2 (PDCD1LG2) gene, glutamate
receptor (ionotropic (N-methyl D-aspartate-like 1A (GRINL1A) gene,
mahogunin (ring finger 1 (MGRN1) gene, syntrophin (beta 2
(dystrophin-associated protein A1 (59 kDa (basic component 2)
(SNTB2) gene, cyclin-dependent kinase 5 (regulatory subunit 1 (p35)
(CDK5R1) gene, glucosidase (alpha; acid (GAA) gene, katanin p60
subunit A-like 2 (KATNAL2) gene, carcinoembryonic antigen-related
cell adhesion molecule 4 (CEACAM4) gene, zinc finger protein 335
(ZNF335) gene, aspartate beta-hydroxylase domain containing 2
(ASPHD2) gene, acidic repeat containing (ACRC) gene,
butyrophilin-like 3/butyrophilin-like 8 (BTNL3, BTLN8) gene,
Moloney leukemia virus 10 homolog (mouse) (MOV10) gene, mediator
complex subunit 12-like (MED12L) gene, kelch-like 6 (Drosophila)
(KLHL6) gene, PDZ and LIM domain 5 (PDLIM5) gene,
UDP-N-acetyl-alpha-D-galactosamine:polypeptide
N-acetylgalactosaminyltransferase 10 (GALNT10) gene, secernin 1
(SCRN1) gene, vesicular (overexpressed in cancer (prosurvival
protein 1 (VOPP1, RP11-289I10.2) gene, FK506 binding protein 9, 63
kDa (FKBP9, FKBP9, FKBP9L, AC091812.2) gene, kinesin family member
27 (KIF27) gene, piwi-like 4 (Drosophila) (PIWIL4) gene,
telomerase-associated protein 1 (TEP1) gene, GTP cyclohydrolase 1,
(GCH1) gene, proline rich 11, (PRR11) gene, cadherin 2, type 1,
N-cadherin (neuronal) (CDH2) gene, protein phosphatase 1B-like
(FLJ40125, AC138534.1) (PPM1N) gene, related RAS viral (r-ras)
oncogene homolog, (RRAS) gene,
dolichyl-diphosphooligosaccharide-protein glycosyltransferase,
(DDOST) gene, anterior pharynx defective 1 homolog A (C. elegans)
(APH1A) gene, tubulin tyrosine ligase (TTL) gene, testis expressed
261, (TEX261) gene, coenzyme Q2 homolog, prenyltransferase (yeast)
(COQ2) gene, FCH and double SH3 domains 1, (FCHSD1) gene,
BCL2-antagonist/killer 1, (BAK1) gene, solute carrier family 25
(mitochondrial carrier; phosphate carrier) member 25, (SLC25A25)
gene, RELT tumor necrosis factor receptor, (RELT) gene, acid
phosphatase 2, lysosomal, (ACP2) gene, TBC1 domain family, member
2B, (TBC1D2B) gene, Fanconi anemia, complementation group A,
(FANCA) gene, solute carrier family 39 (metal ion transporter)
member 11, (SLC39A11) gene.
3. A method according to claim 1, comprising: comparing the level
of at least one IRC marker transcript to the level of a
corresponding IRC marker transcript, wherein the IRC marker
transcript is selected from the group consisting of: (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 80% (or at least 81% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,
247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,
273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,
325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,
351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,
377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401,
403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427,
429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453,
455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,
481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505,
507, 509, 511, 513 or 515, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,
176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,
202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,
306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,
332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,
358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,
410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,
436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,
462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,
488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,
514 or 516; (c) a polynucleotide comprising a nucleotide sequence
that encodes a polypeptide that shares at least 80% (or at least
81% to at least 99% and all integer percentages in between)
sequence similarity or identity with at least a portion of the
sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,
220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,
272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,
324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,
350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,
376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,
402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,
454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,
480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,
506, 508, 510, 512, 514 or 516; (d) a polynucleotide expression
product comprising a nucleotide sequence that hybridizes to the
sequence of (a), (b), (c) or a complement thereof, under at least
medium or high stringency conditions.
4. A method according to claim 1, comprising: comparing the level
of at least one IRC marker polypeptide to the level of a
corresponding IRC marker polypeptide, wherein the IRC marker
polypeptide is selected from the group consisting of: (i) a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,
176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,
202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,
306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,
332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,
358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,
410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,
436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,
462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,
488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,
514 or 516; and (ii) a polypeptide comprising an amino acid
sequence that shares at least 80% (or at least 81% to at least 99%
and all integer percentages in between) sequence similarity or
identity with the sequence set forth in any one of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,
262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,
314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,
340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,
366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,
444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,
470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514 or 516.
5. A method according to any one of claims 1 to 4, comprising: (1)
measuring in a biological sample obtained from the subject the
level of the at least one IRC marker expression product and (2)
comparing the measured level of each IRC marker expression product
to the level of a corresponding IRC marker expression product in a
reference sample obtained from the at least one control
subject.
6. A method according to any one of claims 1 to 5, comprising:
assessing whether the subject has, or is at risk of developing, one
of the plurality of conditions when the measured level of the or
each IRC marker expression product is different than the measured
level of the or each corresponding IRC marker expression
product.
7. A method according to claim 6, wherein the level of an
individual IRC marker expression product is at least 110% of the
level of an individual corresponding IRC expression product.
8. A method according to claim 6, wherein the level of an
individual IRC marker expression product is no more than about 95%
of the level of an individual corresponding IRC expression
product.
9. A method according to any one of claims 1 to 6 or 8, wherein the
presence or risk of development of sepsis is determined by
detecting in the subject a decrease in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 IRC marker expression
products from a multi-transcript-producing gene selected from the
group consisting of KIF27, OTX1, CDK5R1, FKBP9, CDH2, ADAM19,
BTNL8/3 and PANX2 (hereafter referred to as "LIST A"), as compared
to the level of a corresponding IRC marker expression product(s) in
a post-surgical inflammation-positive control subject.
10. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of post-surgical inflammation is
determined by detecting in the subject an increase in the level of
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 IRC marker
expression product(s) from at least one multi-transcript-producing
gene selected from the group consisting of: KIF27, OTX1, CDK5R1,
FKBP9, CDH2, ADAM19, BTNL8/3 and PANX2 (i.e., LIST A), as compared
to the level of a corresponding IRC marker expression product in a
sepsis control subject.
11. A method according to claim 9 or claim 10, wherein the KIF27
IRC marker expression product comprises a nucleotide sequence
corresponding to KIF27 exons 4 and 7 or an amino acid sequence
encoded by that exon.
12. A method according to claim 11, wherein the KIF27 IRC marker
expression product is a KIF27 IRC marker transcript as set forth in
any one of SEQ ID NO: 1, 3, 5, 7, or 9.
13. A method according to claim 11, wherein the KIF271 IRC marker
expression product is a KIF27 IRC marker polypeptide as set forth
in any one of SEQ ID NO: 2, 4, 6, 8, or 10.
14. A method according to claim 9 or claim 10, wherein the OTX1 IRC
marker expression product comprises a nucleotide sequence
corresponding to OTX1 exon 5 or an amino acid sequence encoded by
that exon.
15. A method according to claim 14, wherein the OTX1 IRC marker
expression product is an OTX1 IRC marker transcript as set forth in
any one of SEQ ID NO: 11 or 13.
16. A method according to claim 14, wherein the OTX1 IRC marker
expression product is an OTX1 IRC marker polypeptide as set forth
in any one of SEQ ID NO:12 or 14.
17. A method according to claim 9 or claim 10, wherein the CDK5R1
IRC marker expression product comprises a nucleotide sequence
corresponding to CDK5R1 exon 2 or an amino acid sequence encoded by
that exon.
18. A method according to claim 17, wherein the CDK5R1 IRC marker
expression product is a CDK5R1 IRC marker transcript as set forth
in any one of SEQ ID NO: 15.
19. A method according to claim 17, wherein the CDK5R1 IRC marker
expression product is a CDK5R1 IRC marker polypeptide as set forth
in any one of SEQ ID NO: 16.
20. A method according to claim 9 or claim 10, wherein the FKBP9
IRC marker expression product comprises a nucleotide sequence
corresponding to FKBP9 exon 10 or an amino acid sequence encoded by
that exon.
21. A method according to claim 20, wherein the IRC marker
expression product is an FKBP9 IRC marker transcript as set forth
in any one of SEQ ID NO: 17.
22. A method according to claim 20, wherein the FKBP9 IRC marker
expression product is an FKBP9 IRC marker polypeptide as set forth
in any one of SEQ ID NO: 18.
23. A method according to claim 9 or claim 10, wherein the CDH2 IRC
marker expression product comprises a nucleotide sequence
corresponding to CDH2 exon 10 or an amino acid sequence encoded by
that exon.
24. A method according to claim 23, wherein the CDH2 IRC marker
expression product is a CDH2 IRC marker transcript as set forth in
any one of SEQ ID NO: 19 and 21.
25. A method according to claim 23, wherein the CDH2 IRC marker
expression product is a CDH2 IRC marker polypeptide as set forth in
any one of SEQ ID NO:19 and 21.
26. A method according to claim 9 or claim 10, wherein the ADAM19
IRC marker expression product comprises a nucleotide sequence
corresponding to ADAM19 exon 10 or an amino acid sequence encoded
by that exon.
27. A method according to claim 26, wherein the ADAM19 IRC marker
expression product is an ADAM 19 IRC marker transcript as set forth
in any one of SEQ NO: 23, 25, 27 and 29.
28. A method according to claim 26, wherein the ADAM19 IRC marker
expression product is an ADAM19 IRC marker polypeptide as set forth
in any one of SEQ ID NO:24, 26, 28 and 30.
29. A method according to claim 9 or claim 10, wherein the BTNL8/3
IRC marker expression product comprises a nucleotide sequence
corresponding to BTNL8/3 exon 6 or an amino acid sequence encoded
by that exon.
30. A method according to claim 29, wherein the BTNL8/3 IRC marker
expression product is a BTNL8/3IRC marker transcript as set forth
in any one of SEQ ID NO: 31, 33, 35, 37, 39 and 41.
31. A method according to claim 29, wherein the BTNL8/3 IRC marker
expression product is a BTNL8/3 IRC marker polypeptide as set forth
in any one of SEQ ID NO: 32, 34, 36, 38, 40 and 42.
32. A method according to claim 9 or claim 10, wherein the PANX2
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from PANX2 exon 1 and exon 2, or
an amino acid sequence encoded by that exon.
33. A method according to claim 32, wherein the PANX2 IRC marker
expression product is a PANX2 IRC transcript as set forth in any
one of SEQ ID NO: 43, 45 or 47.
34. A method according to claim 32, wherein the PANX2 IRC marker
expression product is a PANX2 IRC polypeptide as set forth in any
one of SEQ ID NO:44, 46 or 48.
35. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of sepsis is determined by
detecting in the subject an increase in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157 or 158 IRC marker expression product(s) from at least one
multi-transcript-producing gene selected from the group consisting
of: PDLIM5, SCRN1, ASPHD2, VOPP1, ACRC, GALNT10, AC1385341, MED12L,
RHBDF2, KLHL6, TEP1, PIWIL6, PRR1, RRAS, TG, ANKDD1A, GABRR2,
MOV10, SLAMF7, PDCDILG2 and GCH1 (hereafter referred to as "LIST
B"), as compared to the level of a corresponding IRC marker
expression product in a post-surgical-positive subject control
subject.
36. A method according to any one of claims 1 to 6, or 8, wherein
the presence or risk of development of post-surgical inflammation
is determined by detecting in the subject a decrease in the level
of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,
51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,
101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,
127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,
140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152,
153, 154, 155, 156, 157 or 158 IRC marker expression product(s)
from at least one multi-transcript-producing gene selected from the
group consisting of PDLIM5, SCRN1, ASPHD2, VOPP1, ACRC, GALNT10,
AC1385341, MED12L, RHBDF2, KLHL6, TEP1, PIWIL6, PRR1, RRAS, TG,
ANKDD1A, GABRR2, MOV10, SLAMF7, PDCDILG2 and GCH1 (i.e., LIST B),
as compared to the level of a corresponding IRC marker expression
product in a sepsis control subject.
37. A method according to claim 35 or claim 36, wherein the PDLIM5
IRC marker expression product comprises a nucleotide sequence
corresponding to PDLIM5 exon 5 or an amino acid sequence encoded by
that exon.
38. A method according to claim 37, wherein the PDLIM5 IRC marker
expression product is a PDLIM5 IRC transcript as set forth in any
one of SEQ ID NO: 49.
39. A method according to claim 37, wherein the PDLIM5 IRC marker
expression product is a PDLIM5 IRC polypeptide as set forth in any
one of SEQ ID NO: 50.
40. A method according to claim 35 or claim 36, wherein the SCRN1
IRC marker expression product comprises a nucleotide sequence
corresponding to SCRN1 exon 5 or an amino acid sequence encoded by
that exon.
41. A method according to claim 40, wherein the SCRN1 IRC marker
expression product is a SCRN1 IRC transcript as set forth in any
one of SEQ ID NO: 51, 53, 55, 57, 59, 61 or 63.
42. A method according to claim 40, wherein the SCRN1 IRC marker
expression product is a SCRN1 IRC polypeptide as set forth in any
one of SEQ ID NO: 52, 54, 56, 58, 60, 62 or 64.
43. A method according to claim 35 or claim 36, wherein the ASPHD2
IRC marker expression product comprises a nucleotide sequence
corresponding to ASPHD2 exon 4 or an amino acid sequence encoded by
that exon.
44. A method according to claim 43, wherein the ASPHD2 IRC marker
expression product is an ASPHD2 IRC transcript as set forth in any
one of SEQ ID NO:65, 67 or 69.
45. A method according to claim 43, wherein the ASPHD2 IRC marker
expression product is an ASPHD2 IRC polypeptide as set forth in any
one of SEQ ID NO:66, 68 or 70.
46. A method according to claim 35 or claim 36, wherein the VOPP1
IRC marker expression product comprises a nucleotide sequence
corresponding to VOPP1 exon 3 or an amino acid sequence encoded by
that exon.
47. A method according to claim 46, wherein the VOPP1 IRC marker
expression product is a VOPP1 IRC transcript as set forth in any
one of SEQ ID NO: 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 or
93.
48. A method according to claim 46, wherein the VOPP1 IRC marker
expression product is a VOPP1 IRC polypeptide as set forth in any
one of SEQ ID NO: 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 or
94.
49. A method according to claim 35 or claim 36, wherein the ACRC
IRC marker expression product comprises a nucleotide sequence
corresponding to ACRC exons 3 and 5 or an amino acid sequence
encoded by that exon.
50. A method according to claim 49, wherein the ACRC IRC marker
expression product is an ACRC IRC transcript as set forth in any
one of SEQ ID NO:95 or 97.
51. A method according to claim 49, wherein the ACRC IRC marker
expression product is an ACRC IRC polypeptide as set forth in any
one of SEQ ID NO:96 or 98.
52. A method according to claim 35 or claim 36, wherein the GALNT10
IRC marker expression product comprises a nucleotide sequence
corresponding to GALNT10 exon 6 or an amino acid sequence encoded
by that exon.
53. A method according to claim 52, wherein the GALNT10 IRC marker
expression product is a GALNT10 IRC transcript as set forth in any
one of SEQ ID NO:99 or 101.
54. A method according to claim 52, wherein the GALNT10 IRC marker
expression product is a GALNT10 IRC polypeptide as set forth in any
one of SEQ ID NO:100 or 102.
55. A method according to claim 35 or claim 36, wherein the PPM1N
IRC marker expression product comprises a nucleotide sequence
corresponding to PPM1N exon 3 or an amino acid sequence encoded by
that exon.
56. A method according to claim 55, wherein the PPM1N IRC marker
expression product is a PPM1N IRC transcript as set forth in any
one of SEQ ID NO: 107, 109, 111, 113, 115, 117, 119, 121 or
123.
57. A method according to claim 55, wherein the PPM1N IRC marker
expression product is a PPM1N IRC polypeptide as set forth in any
one of SEQ ID NO: 108, 110, 112, 114, 116, 118, 120, 122, or
124.
58. A method according to claim 35 or claim 36, wherein the MED12L
IRC marker expression product comprises a nucleotide sequence
corresponding to MED12L exon 17 or an amino acid sequence encoded
by that exon.
59. A method according to claim 58, wherein the MED12L IRC marker
expression product is a MED12L IRC transcript as set forth in any
one of SEQ ID NO: 125 or 127.
60. A method according to claim 58, wherein the MED12L IRC marker
expression product is a MED12L IRC polypeptide as set forth in any
one of SEQ ID NO:126 or 128.
61. A method according to claim 35 or claim 36, wherein the RHBDF2
IRC marker expression product comprises a nucleotide sequence
corresponding to RHBDF2 exons 6, 9, 10, 11, 14, 17, 18 or 19 or an
amino acid sequence encoded by that exon.
62. A method according to claim 61, wherein the RHBDF2 IRC marker
expression product is an RHBDF2 IRC transcript as set forth in any
one of SEQ ID NO: 129, 131 or 133.
63. A method according to claim 61, wherein the RHBDF2 IRC marker
expression product is an RHBDF2 IRC polypeptide as set forth in any
one of SEQ ID NO:130, 132 or 134.
64. A method according to claim 35 or claim 36, wherein the KLHL6
IRC marker expression product comprises a nucleotide sequence
corresponding to KLHL6 exon 7 or an amino acid sequence encoded by
that exon.
65. A method according to claim 64, wherein the KLHL6 IRC marker
expression product is a KLHL6 IRC transcript as set forth in any
one of SEQ ID NO: 135.
66. A method according to claim 64, wherein the KLHL6 IRC marker
expression product is a KLHL6 IRC polypeptide as set forth in any
one of SEQ ID NO:136.
67. A method according to claim 35 or claim 36, wherein the TEP1
IRC marker expression product comprises a nucleotide sequence
corresponding to TEP1 exon 49 or an amino acid sequence encoded by
that exon.
68. A method according to claim 67, wherein the TEP1 IRC marker
expression product is a TEP1 IRC transcript as set forth in any one
of SEQ ID NO: 137 or 139.
69. A method according to claim 67, wherein the TEP1 IRC marker
expression product is a TEP1 IRC polypeptide as set forth in any
one of SEQ ID NO:138 or 140.
70. A method according to claim 35 or claim 36, wherein the PIWIL4
IRC marker expression product comprises a nucleotide sequence
corresponding to PIWIL4 exons 2 and 14 or an amino acid sequence
encoded by that exon.
71. A method according to claim 70, wherein the PIWIL4 IRC marker
expression product is a PIWIL4 IRC transcript as set forth in any
one of SEQ ID NO: 141 or 143.
72. A method according to claim 70, wherein the PIWIL4 IRC marker
expression product is a PIWIL4 IRC polypeptide as set forth in any
one of SEQ ID NO:142 or 144.
73. A method according to claim 35 or claim 36, wherein the PRR11
IRC marker expression product comprises a nucleotide sequence
corresponding to PRR11 exons 4 and 5 or an amino acid sequence
encoded by that exon.
74. A method according to claim 73, wherein the PRR11 IRC marker
expression product is a PRR11 IRC transcript as set forth in any
one of SEQ ID NO: 145.
75. A method according to claim 73, wherein the PRR11 IRC marker
expression product is a PRR11 IRC polypeptide as set forth in any
one of SEQ ID NO:146.
76. A method according to claim 35 or claim 36, wherein the RRAS
IRC marker expression product comprises a nucleotide sequence
corresponding to RRAS exon 1 or an amino acid sequence encoded by
that exon.
77. A method according to claim 76, wherein the RRAS IRC marker
expression product is an RRAS IRC transcript as set forth in any
one of SEQ ID NO: 147.
78. A method according to claim 76, wherein the RRAS IRC marker
expression product is an RRAS IRC polypeptide as set forth in any
one of SEQ ID NO:148.
79. A method according to claim 35 or claim 36, wherein the TG IRC
marker expression product comprises a nucleotide sequence
corresponding to TG exon 6 or an amino acid sequence encoded by
that exon.
80. A method according to claim 79, wherein the TG IRC marker
expression product is a TG IRC transcript as set forth in any one
of SEQ ID NO: 149 or 151.
81. A method according to claim 79, wherein the TG IRC marker
expression product is a TG IRC polypeptide as set forth in any one
of SEQ ID NO:150 or 152.
82. A method according to claim 35 or claim 36, wherein the ANKDD1A
IRC marker expression product comprises a nucleotide sequence
corresponding to ANKDD1A exon 7 or an amino acid sequence encoded
by that exon.
83. A method according to claim 82, wherein the ANKDD1A IRC marker
expression product is an ANKDD1A IRC transcript as set forth in any
one of SEQ ID NO: 153, 155, 157, 159 or 161.
84. A method according to claim 82, wherein the ANKDD1A IRC marker
expression product is an ANKDD1A IRC polypeptide as set forth in
any one of SEQ ID NO:154, 156, 158, 160 or 162.
85. A method according to claim 35 or claim 36, wherein the GABRR2
IRC marker expression product comprises a nucleotide sequence
corresponding to GABRR2 exons 7, 8 or 9 or an amino acid sequence
encoded by that exon.
86. A method according to claim 85, wherein the GABRR2 IRC marker
expression product is an GABRR2 IRC transcript as set forth in any
one of SEQ ID NO: 163 or 165.
87. A method according to claim 85, wherein the GABRR2 IRC marker
expression product is an GABRR2 IRC polypeptide as set forth in any
one of SEQ ID NO:164 or 166.
88. A method according to claim 35 or claim 36, wherein the MOV10
IRC marker expression product comprises a nucleotide sequence
corresponding to MOV10 exon 6 or an amino acid sequence encoded by
that exon.
89. A method according to claim 88, wherein the MOV10 IRC marker
expression product is a MOV10 IRC transcript as set forth in any
one of SEQ ID NO: 167, 169, 171, 173, 175 or 177.
90. A method according to claim 85, wherein the MOV10 IRC marker
expression product is a MOV10 IRC polypeptide as set forth in any
one of SEQ ID NO:168, 170, 172, 174, 176 or 178.
91. A method according to claim 35 or claim 36, wherein the SLAMF7
IRC marker expression product comprises a nucleotide sequence
corresponding to SLAMF7 exons 2, 3, 4 or 5 or an amino acid
sequence encoded by that exon.
92. A method according to claim 91, wherein the SLAMF7 IRC marker
expression product is a SLAMF7 IRC transcript as set forth in any
one of SEQ ID NO: 179, 181, 183, 185, 187, 189, 191 or 193.
93. A method according to claim 91, wherein the SLAMF7 IRC marker
expression product is a SLAMF7 IRC polypeptide as set forth in any
one of SEQ ID NO:180, 182, 184, 186, 188, 190, 192 or 194.
94. A method according to claim 35 or claim 36, wherein the
PDCD1LG2 IRC marker expression product comprises a nucleotide
sequence corresponding to PDCD1LG2 exons 1 or 2 or an amino acid
sequence encoded by that exon.
95. A method according to claim 94, wherein the PDCD1LG2 IRC marker
expression product is a PDCD1LG2 IRC transcript as set forth in any
one of SEQ ID NO:195 or 197.
96. A method according to claim 94, wherein the PDCD1LG2 IRC marker
expression product is a PDCD1LG2 IRC polypeptide as set forth in
any one of SEQ ID NO: 196 or 198.
97. A method according to claim 35 or claim 36, wherein the GCH1 RC
marker expression product comprises a nucleotide sequence
corresponding to GCH1 exon 2 or an amino acid sequence encoded by
that exon.
98. A method according to claim 97, wherein the GCH1 IRC marker
expression product is a GCH1 IRC transcript as set forth in any one
of SEQ ID NO:199, 201, 203 or 205.
99. A method according to claim 97, wherein the GCH1 IRC marker
expression product is a GCH1 IRC polypeptide as set forth in any
one of SEQ ID NO: 200, 202, 204 or 206.
100. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of sepsis is determined by
detecting in the subject an increase in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155 or
156 IRC marker expression product(s) from at least one
multi-transcript-producing gene selected from the group consisting
of: RELT, ACP2, FCHSD1, CLPB, SLC39A1, TBC1D2B, APH1A, DDOST, BAK1,
SLC25A25A, COQ2, FANCA, PIWIL4, ZNF335, TEX261, GABRR2, VOPP1, TTL,
CES2, GALNT10, CQORF91, AMBRA1 and SCRN1 (hereafter referred to as
"LIST C"), as compared to the level of a corresponding IRC marker
expression product in an inSIRS-positive control subject.
101. A method according to any one of claims 1 to 6, or 8, wherein
the presence or risk of development of inSIRS is determined by
detecting in the subject a decrease in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,
104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,
117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,
143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155 or
156 IRC marker expression(s) product from at least one
multi-transcript-producing gene selected from the group consisting
of: RELT, ACP2, FCHSD1, CLPB, SLC39A1, TBC1D2B, APH1A, DDOST, BAK1,
SLC25A25A, COQ2, FANCA, PIWIL4, ZNF335, TEX261, GABRR2, VOPP1, TTL,
CES2, GALNT10, CQORF91, AMBRA1 and SCRN1 (i.e., LIST C), as
compared to the level of the corresponding IRC marker expression
product in a sepsis-positive control subject.
102. A method according to claim 100 or claim 101, wherein the RELT
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from RELT exon 4, or an amino
acid sequence encoded by that exon.
103. A method according to claim 102, wherein the RELT IRC marker
expression product is a RELT IRC transcript as set forth in any one
of SEQ ID NO: 207 or 209.
104. A method according to claim 102, wherein the RELT IRC marker
expression product is a RELT IRC polypeptide as set forth in any
one of SEQ ID NO: 208 or 210.
105. A method according to claim 100 or claim 101, wherein the ACP2
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from ACP2 exon 7, or an amino
acid sequence encoded by that exon.
106. A method according to claim 105, wherein the ACP2 IRC marker
expression product is a ACP2 IRC transcript as set forth in any one
of SEQ ID NO: 211.
107. A method according to claim 105, wherein the ACP2 IRC marker
expression product is a ACP2 IRC polypeptide as set forth in any
one of SEQ ID NO: 212.
108. A method according to claim 100 or claim 101, wherein the
FCHSD1 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from FCHSD1 exon 14, or
an amino acid sequence encoded by that exon.
109. A method according to claim 108, wherein the FCHSD1 IRC marker
expression product is a FCHSD1 IRC transcript as set forth in any
one of SEQ ID NO: 213 or 215.
110. A method according to claim 108, wherein the FCHSD1 IRC marker
expression product is a FCHSD1 IRC polypeptide as set forth in any
one of SEQ ID NO: 214 or 216.
111. A method according to claim 100 or claim 101, wherein the CLPB
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from CLPB exon 10, or an amino
acid sequence encoded by that exon.
112. A method according to claim 111, wherein the CLPB IRC marker
expression product is a CLPB IRC transcript as set forth in any one
of SEQ ID NO: 217, 219 or 221.
113. A method according to claim 111, wherein the CLPB IRC marker
expression product is a CLPB IRC polypeptide as set forth in any
one of SEQ ID NO: 218, 220 or 222.
114. A method according to claim 100 or claim 101, wherein the
SLC39A11 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from SLC39A11 exon 2, or
an amino acid sequence encoded by that exon.
115. A method according to claim 114, wherein the SLC39A11 IRC
marker expression product is a SLC39A11 IRC transcript as set forth
in any one of SEQ ID NO: 223.
116. A method according to claim 114, wherein the SLC39A11 IRC
marker expression product is a SLC39A11 IRC polypeptide as set
forth in any one of SEQ ID NO: 224.
117. A method according to claim 100 or claim 101, wherein the
TBC1D2B IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from TBC1D2B exon 13, or
an amino acid sequence encoded by that exon.
118. A method according to claim 117, wherein the TBC1D2B IRC
marker expression product is a TBC1D2B IRC transcript as set forth
in any one of SEQ ID NO: 225, 227 or 229.
119. A method according to claim 117, wherein the TBC1D2B IRC
marker expression product is a TBC1D2B IRC polypeptide as set forth
in any one of SEQ ID NO: 226, 228 or 230.
120. A method according to claim 100 or claim 101, wherein the
APH1A IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from APH1A exon 1, or an amino
acid sequence encoded by that exon.
121. A method according to claim 120, wherein the APH1A IRC marker
expression product is an APH1A IRC transcript as set forth in any
one of SEQ ID NO: 231, 233, 235, 237, 239 or 241.
122. A method according to claim 120, wherein the APH1A IRC marker
expression product is a APH1A IRC polypeptide as set forth in any
one of SEQ ID NO: 232, 234, 236, 238, 240 or 242.
123. A method according to claim 100 or claim 101, wherein the
DDOST IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from DDOST exon 2, or an amino
acid sequence encoded by that exon.
124. A method according to claim 123, wherein the DDOST IRC marker
expression product is a DDOST IRC transcript as set forth in any
one of SEQ ID NO: 243.
125. A method according to claim 123, wherein the DDOST IRC marker
expression product is a DDOST IRC polypeptide as set forth in any
one of SEQ ID NO: 244.
126. A method according to claim 100 or claim 101, wherein the BAK1
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from BAK1 exon 7, or an amino
acid sequence encoded by that exon.
127. A method according to claim 126, wherein the BAK1 IRC marker
expression product is a BAK1 IRC transcript as set forth in any one
of SEQ ID NO: 245 or 247.
128. A method according to claim 126, wherein the BAK1 IRC marker
expression product is a BAK1 IRC polypeptide as set forth in any
one of SEQ ID NO: 246 or 248.
129. A method according to claim 100 or claim 101, wherein the
SLC25A25A IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from SLC25A25A exon 10,
or an amino acid sequence encoded by that exon.
130. A method according to claim 129, wherein the SLC25A25A IRC
marker expression product is an SLC25A25A IRC transcript as set
forth in any one of SEQ ID NO: 249, 251, 253, 255, 257, 259 or
261.
131. A method according to claim 129, wherein the SLC25A25A IRC
marker expression product is an SLC25A25A IRC polypeptide as set
forth in any one of SEQ ID NO: 250, 252, 254, 256, 258, 260 or
262.
132. A method according to claim 100 or claim 101, wherein the COQ2
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from COQ2 exon 1, or an amino
acid sequence encoded by that exon.
133. A method according to claim 132, wherein the COQ2 IRC marker
expression product is a COQ2 IRC transcript as set forth in any one
of SEQ ID NO: 263, 265 or 267.
134. A method according to claim 132, wherein the COQ2 IRC marker
expression product is a COQ2 IRC polypeptide as set forth in any
one of SEQ ID NO: 264, 266 or 268.
135. A method according to claim 100 or claim 101, wherein the
FANCA IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from FANCA exon 35, or an amino
acid sequence encoded by that exon.
136. A method according to claim 135, wherein the FANCA IRC marker
expression product is a FANCA IRC transcript as set forth in any
one of SEQ ID NO: 269 or 271.
137. A method according to claim 135, wherein the FANCA IRC marker
expression product is a FANCA IRC polypeptide as set forth in any
one of SEQ ID NO: 270 or 272.
138. A method according to claim 100 or claim 101, wherein the
PIWIL4 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from PIWIL4 exons 2, 14,
or an amino acid sequence encoded by that exon.
139. A method according to claim 138, wherein the PIWIL4 IRC marker
expression product is a PIWIL4 IRC transcript as set forth in any
one of SEQ ID NO: 273 or 275.
140. A method according to claim 138, wherein the PIWIL4 IRC marker
expression product is a PIWIL4 IRC polypeptide as set forth in any
one of SEQ ID NO: 274 or 276.
141. A method according to claim 100 or claim 101, wherein the
ZNF335 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from ZNF335 exon 5, or
an amino acid sequence encoded by that exon.
142. A method according to claim 141, wherein the ZNF335 IRC marker
expression product is a ZNF335 IRC transcript as set forth in any
one of SEQ ID NO: 277, 279 or 281.
143. A method according to claim 141, wherein the ZNF335 IRC marker
expression product is a ZNF335 IRC polypeptide as set forth in any
one of SEQ ID NO: 278, 280 or 282.
144. A method according to claim 100 or claim 101, wherein the
TEX261 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from TEX261 exon 3, or
an amino acid sequence encoded by that exon.
145. A method according to claim 144, wherein the TEX261 IRC marker
expression product is a TEX261 IRC transcript as set forth in any
one of SEQ ID NO: 283 or 285.
146. A method according to claim 144, wherein the TEX261 IRC marker
expression product is a TEX261 IRC polypeptide as set forth in any
one of SEQ ID NO: 284 or 286.
147. A method according to claim 100 or claim 101, wherein the
GABRR2 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from GABRR2 exons 7, 8,
9 or an amino acid sequence encoded by that exon.
148. A method according to claim 147, wherein the GABRR2 IRC marker
expression product is a GABRR2 IRC transcript as set forth in any
one of SEQ ID NO: 287 or 289.
149. A method according to claim 147, wherein the GABRR2 IRC marker
expression product is a GABRR2 IRC polypeptide as set forth in any
one of SEQ ID NO: 288 or 290.
150. A method according to claim 100 or claim 101, wherein the
VOPP1 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from VOPP1 exon 3 or an amino
acid sequence encoded by that exon.
151. A method according to claim 150, wherein the VOPP1 IRC marker
expression product is a VOPP1 IRC transcript as set forth in any
one of SEQ ID NO: 291, 293, 295, 297, 299, 301, 303, 305, 307, 309,
311 or 313.
152. A method according to claim 150, wherein the VOPP1 IRC marker
expression product is a VOPP1 IRC polypeptide as set forth in any
one of SEQ ID NO: 292, 294, 296, 298, 300, 302, 304, 306, 308, 310,
312 or 314.
153. A method according to claim 100 or claim 101, wherein the TTL
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from TTL exon 7 or an amino acid
sequence encoded by that exon.
154. A method according to claim 153, wherein the TTL IRC marker
expression product is a TTL IRC transcript as set forth in any one
of SEQ ID NO: 315.
155. A method according to claim 153, wherein the TTL IRC marker
expression product is a TTL IRC polypeptide as set forth in any one
of SEQ ID NO: 316.
156. A method according to claim 100 or claim 101, wherein the CES2
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from CES2 exon 1 or an amino acid
sequence encoded by that exon.
157. A method according to claim 156, wherein the CES2 IRC marker
expression product is a CES2 IRC transcript as set forth in any one
of SEQ ID NO: 317 or 319.
158. A method according to claim 156, wherein the CES2 IRC marker
expression product is a CES2 IRC polypeptide as set forth in any
one of SEQ ID NO: 318 or 320.
159. A method according to claim 100 or claim 101, wherein the
GALNT10 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from GALNT10 exon 6 or
an amino acid sequence encoded by that exon.
160. A method according to claim 159, wherein the GALNT10 IRC
marker expression product is a GALNT10 IRC transcript as set forth
in any one of SEQ ID NO: 321 or 323.
161. A method according to claim 159, wherein the GALNT10 IRC
marker expression product is a GALNT10 IRC polypeptide as set forth
in any one of SEQ ID NO: 322 or 324.
162. A method according to claim 100 or claim 101, wherein the
C1orf91 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from C1orf91 exon 2 or
an amino acid sequence encoded by that exon.
163. A method according to claim 162, wherein the C1orf91 IRC
marker expression product is a C1orf91 IRC transcript as set forth
in any one of SEQ ID NO: 325, 327, 329, 331, 333 or 335.
164. A method according to claim 162, wherein the C1orf91 IRC
marker expression product is a C1orf91 IRC polypeptide as set forth
in any one of SEQ ID NO: 326, 328, 330, 332, 334 or 336.
165. A method according to claim 100 or claim 101, wherein the
AMBRA1 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from AMBRA1 exons 2, 4
or an amino acid sequence encoded by that exon.
166. A method according to claim 165, wherein the AMBRA1 IRC marker
expression product is a AMBRA1 IRC transcript as set forth in any
one of SEQ ID NO: 337, 339, 341, 343, 345 or 347.
167. A method according to claim 165, wherein the AMBRA1 IRC marker
expression product is a AMBRA1 IRC polypeptide as set forth in any
one of SEQ ID NO: 338, 340, 342, 344, 346 or 348.
168. A method according to claim 100 or claim 101, wherein the SCRN
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from SCRN exon 5 or an amino acid
sequence encoded by that exon.
169. A method according to claim 168, wherein the SCRN IRC marker
expression product is a SCRN IRC transcript as set forth in any one
of SEQ ID NO: 349, 3512, 353, 355, 357, 359 or 361.
170. A method according to claim 168, wherein the SCRN IRC marker
expression product is a SCRN IRC polypeptide as set forth in any
one of SEQ ID NO: 350, 352, 354, 356, 358, 360 or 362.
171. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of inSIRS is determined by
detecting in the subject a increase in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
IRC marker expression(s) product from at least one
multi-transcript-producing gene selected from the group consisting
of: GRINL1A and KATLNAL2 (i.e., LIST D), as compared to the level
of the corresponding IRC marker expression product in a
sepsis-positive control subject.
172. A method according to any one of claims 1 to 6, or 8, wherein
the presence or risk of development of sepsis is determined by
detecting in the subject an decrease in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
IRC marker expression product(s) from at least one
multi-transcript-producing gene selected from the group consisting
of GRINL1A and KATLNAL2 (hereafter referred to as "LIST D"), as
compared to the level of a corresponding IRC marker expression
product in an inSIRS-positive control subject.
173. A method according to claim 171 or claim 172, wherein the
GRINL1A marker expression product comprises a nucleotide sequence
corresponding to an exon selected from GRINL1A exon 5 or an amino
acid sequence encoded by that exon.
174. A method according to claim 173, wherein the GRINL1A IRC
marker expression product is a GRINL1A IRC transcript as set forth
in any one of SEQ ID NO: 363, 365, 367, 369, 371, 373, 375 or
377.
175. A method according to claim 173, wherein the GRINL1A IRC
marker expression product is a GRINL1A IRC polypeptide as set forth
in any one of SEQ ID NO: 364, 366, 368, 370, 372, 374, 376 or
378.
176. A method according to claim 171 or claim 172, wherein the
KATNAL2 marker expression product comprises a nucleotide sequence
corresponding to an exon selected from KATNAL2 exon 3 or an amino
acid sequence encoded by that exon.
177. A method according to claim 176, wherein the KATNAL2 IRC
marker expression product is a KATNAL2 IRC transcript as set forth
in any one of SEQ ID NO: 379 or 381.
178. A method according to claim 176, wherein the KATNAL2 IRC
marker expression product is a KATNAL2 IRC polypeptide as set forth
in any one of SEQ ID NO: 380 or 382.
179. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of inSIRS is determined by
detecting in the subject an increase in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37
or 38 IRC marker expression products) from at least one
multi-transcript-producing gene selected from the group consisting
of: KATLNAL2, GRINL1A, ACRC, TG and ASPHD2 (hereafter referred to
as "LIST E"), as compared to the level of a corresponding IRC
marker expression product in an post-surgical inflammation-positive
control subject.
180. A method according to any one of claims 1 to 6, or 8, wherein
the presence or risk of development of post-surgical inflammation
is determined by detecting in the subject a decrease in the level
of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37 or 38 IRC marker expression(s) product from at least
one multi-transcript-producing gene selected from the group
consisting of: KATLNAL2, GRINL1A, ACRC, TG and ASPHD2 (i.e., LIST
E), as compared to the level of the corresponding IRC marker
expression product in a inSIRS-positive control subject.
181. A method according to claim 179 or claim 180, wherein the
KATLNAL2 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from KATLNAL2 exon 3, or
an amino acid sequence encoded by that exon.
182. A method according to claim 181, wherein the KATLNAL2 IRC
marker expression product is a KATLNAL2 IRC transcript as set forth
in any one of SEQ ID NO: 387 or 389.
183. A method according to claim 181, wherein the KATLNAL2 IRC
marker expression product is a KATLNAL2 IRC polypeptide as set
forth in any one of SEQ ID NO: 388 or 390.
184. A method according to claim 179 or claim 180, wherein the
GRINL1A IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from GRINL1A exon 5, or
an amino acid sequence encoded by that exon.
185. A method according to claim 184, wherein the GRINL1A IRC
marker expression product is a GRINL1A IRC transcript as set forth
in any one of SEQ ID NO: 391, 393, 395, 397, 399, 401, 403 or
405.
186. A method according to claim 184, wherein the GRINL1A IRC
marker expression product is a GRINL1 A IRC polypeptide as set
forth in any one of SEQ ID NO: 392, 394, 396, 398, 400, 402, 404 or
406.
187. A method according to claim 179 or claim 180, wherein the ACRC
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from ACRC exon 3, 5 or an amino
acid sequence encoded by that exon.
188. A method according to claim 187, wherein the ACRC IRC marker
expression product is a ACRC IRC transcript as set forth in any one
of SEQ ID NO:407 or 409.
189. A method according to claim 187, wherein the ACRC IRC marker
expression product is a ACRC IRC polypeptide as set forth in any
one of SEQ ID NO: 408 or 410.
190. A method according to claim 179 or claim 180, wherein the TG
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from TG exon 6 or an amino acid
sequence encoded by that exon.
191. A method according to claim 190, wherein the TG IRC marker
expression product is a TG IRC transcript as set forth in any one
of SEQ ID NO:411 or 413.
192. A method according to claim 190, wherein the TG IRC marker
expression product is a TG IRC polypeptide as set forth in any one
of SEQ ID NO: 412 or 414.
193. A method according to claim 179 or claim 180, wherein the
ASPHD2 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from ASPHD2 exon 4 or an
amino acid sequence encoded by that exon.
194. A method according to claim 193, wherein the ASPHD2 IRC marker
expression product is an ASPHD2 IRC transcript as set forth in any
one of SEQ ID NO:415, 417 or 419.
195. A method according to claim 193, wherein the ASPHD2 IRC marker
expression product is an ASPHD2 IRC polypeptide as set forth in any
one of SEQ ID NO: 416, 418 or 420.
196. A method according to any one of claims 1 to 7, wherein the
presence or risk of development of post-surgical inflammation is
determined by detecting in the subject an increase in the level of
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 IRC marker
expression(s) product from at least one multi-transcript-producing
gene selected from the group consisting of: CUL7, BTNL8/3, PANX2,
C1orf91, ZNF335, MGRN1, GAA, CDK5R1, SNTB2, CLPB, ADMA19, SLC36A1,
FKBP9, NDST1, HIPK2 and CEACAM4 (i.e., LIST F), as compared to the
level of the corresponding IRC marker expression product in a
inSIRS-positive control subject.
197. A method according to any one of claims 1 to 6, or 8, wherein
the presence or risk of development of inSIRS is determined by
detecting in the subject a decrease in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95 or 96 IRC marker expression product(s)
from at least one multi-transcript-producing gene selected from the
group consisting of CUL7, BTNL8/3, PANX2, C1orf91, ZNF335, MGRN1,
GAA, CDK5R1, SNTB2, CLPB, ADMA19, SLC36A1, FKBP9, NDST1, HIPK2 and
CEACAM4 (hereafter referred to as "LIST F"), as compared to the
level of a corresponding IRC marker expression product in an
post-surgical inflammation-positive control subject.
198. A method according to claim 196 or claim 197, wherein the CUL7
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from CUL7 exon 5 or an amino acid
sequence encoded by that exon.
199. A method according to claim 198, wherein the CUL7 IRC marker
expression product is a CUL7 IRC transcript as set forth in any one
of SEQ ID NO: 421.
200. A method according to claim 198, wherein the CUL7 IRC marker
expression product is a CUL7 IRC polypeptide as set forth in any
one of SEQ ID NO: 422.
201. A method according to claim 196 or claim 197, wherein the
BTNL8/3 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from BTNL8/3 exon 6 or
an amino acid sequence encoded by that exon.
202. A method according to claim 201, wherein the BTNL8 IRC marker
expression product is a BTNL8/3 IRC transcript as set forth in any
one of SEQ ID NO: 423, 425, 427, 429, 431 or 433.
203. A method according to claim 201, wherein the BTNL8 IRC marker
expression product is a BTNL8/3 IRC polypeptide as set forth in any
one of SEQ ID NO: 424, 426, 428, 430, 432 or 434.
204. A method according to claim 196 or claim 197, wherein the
PANX2 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from PANX2 exon 1, 2 or an amino
acid sequence encoded by that exon.
205. A method according to claim 204, wherein the PANX2 IRC marker
expression product is a PANX2 IRC transcript as set forth in any
one of SEQ ID NO: 435, 437 or 439.
206. A method according to claim 204, wherein the PANX2 IRC marker
expression product is a PANX2 IRC polypeptide as set forth in any
one of SEQ ID NO: 436, 438 or 440.
207. A method according to claim 196 or claim 197, wherein the
C1orf91 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from C1orf91 exon 2 or
an amino acid sequence encoded by that exon.
208. A method according to claim 207, wherein the C1orf91 IRC
marker expression product is a C1orf91 IRC transcript as set forth
in any one of SEQ ID NO: 441, 443, 445, 447, 449 or 451.
209. A method according to claim 207, wherein the C1orf91 IRC
marker expression product is a C1orf91 IRC polypeptide as set forth
in any one of SEQ ID NO: 442, 444, 446, 448, 450 or 452.
210. A method according to claim 196 or claim 197, wherein the
ZNF335 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from ZNF335 exon 5 or an
amino acid sequence encoded by that exon.
211. A method according to claim 210, wherein the ZNF335 IRC marker
expression product is a ZNF335 IRC transcript as set forth in any
one of SEQ ID NO: 453, 455 or 457.
212. A method according to claim 210, wherein the ZNF335 IRC marker
expression product is a ZNF335 IRC polypeptide as set forth in any
one of SEQ ID NO: 454, 456 or 458.
213. A method according to claim 196 or claim 197, wherein the
MGRN1 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from MGRN1 exon 4 or an amino
acid sequence encoded by that exon.
214. A method according to claim 213, wherein the MGRN1 IRC marker
expression product is a MGRN1 IRC transcript as set forth in any
one of SEQ ID NO: 459, 461 or 463.
215. A method according to claim 213, wherein the MGRN1 IRC marker
expression product is a MGRN1 IRC polypeptide as set forth in any
one of SEQ ID NO:460, 462 or 464.
216. A method according to claim 196 or claim 197, wherein the GAA
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from GAA exon 3 or an amino acid
sequence encoded by that exon.
217. A method according to claim 216, wherein the GAA IRC marker
expression product is a GAA IRC transcript as set forth in any one
of SEQ ID NO: 465, 467 or 469.
218. A method according to claim 216, wherein the GAA IRC marker
expression product is a GAA IRC polypeptide as set forth in any one
of SEQ ID NO:466, 468 or 470.
219. A method according to claim 196 or claim 197, wherein the
CDK5R1 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from CDK5R1 exon 2 or an
amino acid sequence encoded by that exon.
220. A method according to claim 219, wherein the CDK5R1 IRC marker
expression product is a CDK5R1 IRC transcript as set forth in any
one of SEQ ID NO: 471.
221. A method according to claim 219, wherein the CDK5R1 IRC marker
expression product is a CDK5R1 IRC polypeptide as set forth in any
one of SEQ ID NO: 472.
222. A method according to claim 196 or claim 197, wherein the
SNTB2 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from SNTB2 exon 4 or an amino
acid sequence encoded by that exon.
223. A method according to claim 222, wherein the SNTB2 IRC marker
expression product is a SNTB2 IRC transcript as set forth in any
one of SEQ ID NO: 473.
224. A method according to claim 222, wherein the SNTB2 IRC marker
expression product is a SNTB2 IRC polypeptide as set forth in any
one of SEQ ID NO: 474.
225. A method according to claim 196 or claim 197, wherein the CLPB
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from CLPB exon 10 or an amino
acid sequence encoded by that exon.
226. A method according to claim 225, wherein the CLPB IRC marker
expression product is a CLPB IRC transcript as set forth in any one
of SEQ ID NO: 475, 477 or 479.
227. A method according to claim 225, wherein the CLPB IRC marker
expression product is a CLPB IRC polypeptide as set forth in any
one of SEQ ID NO: 478 or 480.
228. A method according to claim 196 or claim 197, wherein the
ADAM19 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from ADAM19 exon 10 or
an amino acid sequence encoded by that exon.
229. A method according to claim 228, wherein the ADAM19 IRC marker
expression product is an ADAM19 IRC transcript as set forth in any
one of SEQ ID NO: 481, 483, 485 or 487.
230. A method according to claim 228, wherein the ADAM19 IRC marker
expression product is an ADAM19 IRC polypeptide as set forth in any
one of SEQ ID NO: 482, 484, 486 or 488.
231. A method according to claim 196 or claim 197, wherein the
SLC36A1 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from SLC36A1 exon 5 or
an amino acid sequence encoded by that exon.
232. A method according to claim 231, wherein the SLC36A1 IRC
marker expression product is a SLC36A1 IRC transcript as set forth
in any one of SEQ ID NO: 489, 491, 493 or 495.
233. A method according to claim 231, wherein the SLC36A1 IRC
marker expression product is a SLC36A1 IRC polypeptide as set forth
in any one of SEQ ID NO: 490, 492, 494 or 496.
234. A method according to claim 196 or claim 197, wherein the
FKBP9 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from FKBP9 exon 10 or an amino
acid sequence encoded by that exon.
235. A method according to claim 234, wherein the FKBP9 IRC marker
expression product is a FKBP9 IRC transcript as set forth in any
one of SEQ ID NO: 497 or 499.
236. A method according to claim 234, wherein the FKBP9 IRC marker
expression product is a FKBP9 IRC polypeptide as set forth in any
one of SEQ ID NO: 498 or 500.
237. A method according to claim 196 or claim 197, wherein the
NDST1 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from NDST1 exon 3 or an amino
acid sequence encoded by that exon.
238. A method according to claim 237, wherein the NDST1 IRC marker
expression product is a NDST1 IRC transcript as set forth in any
one of SEQ ID NO: 501 or 503.
239. A method according to claim 237, wherein the NDST1 IRC marker
expression product is a NDST1 IRC polypeptide as set forth in any
one of SEQ ID NO: 502 or 504.
240. A method according to claim 196 or claim 197, wherein the
HIPK2 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from HIPK2 exon 11 or an amino
acid sequence encoded by that exon.
241. A method according to claim 240, wherein the HIPK2 IRC marker
expression product is a HIPK2 IRC transcript as set forth in any
one of SEQ ID NO: 505, 507, 509 or 511.
242. A method according to claim 240, wherein the HIPK2 IRC marker
expression product is a HIPK2 IRC polypeptide as set forth in any
one of SEQ ID NO: 506, 508, 510 or 512.
243. A method according to claim 196 or claim 197, wherein the
CEACAM4 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from CEACAM4 exon 5, 7,
23 or an amino acid sequence encoded by that exon.
244. A method according to claim 243, wherein the CEACAM4 IRC
marker expression product is a CEACAM4 IRC transcript as set forth
in any one of SEQ ID NO: 513 or 515.
245. A method according to claim 243, wherein the CEACAM4 IRC
marker expression product is a CEACAM4 IRC polypeptide as set forth
in any one of SEQ ID NO: 514 or 516.
246. A method according to any one of claims 1 to 245, comprising
detecting the level of at least one IRC marker expression product
from two or more of LISTS A, B, C, D, E and F.
247. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B.
248. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST C.
249. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST D.
250. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST E.
251. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST F.
252. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C.
253. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST D.
254. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST E.
255. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST F.
256. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST D.
257. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST E.
258. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST F.
259. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST D and
the level of at least one other IRC marker expression product from
LIST E.
260. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST D and
the level of at least one other IRC marker expression product from
LIST F.
261. A method according to claim 246, comprising detecting the
level of at least one IRC marker expression product from LIST E and
the level of at least one other IRC marker expression product from
LIST F.
262. A method according to any one of claims 1 to 245, comprising
detecting the level of at least one IRC marker expression product
from each of three lists selected from LISTS A, B, C, D, E and
F.
263. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C.
264. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST D.
265. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST E.
266. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST F.
267. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D.
268. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST E.
269. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST F.
270. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E.
271. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST F.
272. A method according to claim 262, comprising detecting the
level of at least one IRC marker expression product from LIST D and
the level of at least one other IRC marker expression product from
LIST E and the level of at least one other IRC marker expression
product from LIST F.
273. A method according to any one of claims 1 to 245, comprising
detecting the level of at least one IRC marker expression product
from each of four lists selected from LISTS A, B, C, D, E and
F.
274. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST D.
275. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST E.
276. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST F.
277. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E.
278. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST F.
279. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E.
280. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST F.
281. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E and the level of at least one other IRC marker
expression product from LIST F.
282. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E.
283. A method according to claim 273, comprising detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E and the level of at least one other RC marker
expression product from LIST F.
284. A method according to any one of claims 1 to 245, comprising
detecting the level of at least one IRC marker expression product
from each of five lists selected from LISTS A, B, C, D, E and
F.
285. A method according to claim 284, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST D and the level of at least one other
IRC marker expression product from LIST E.
286. A method according to claim 284, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST D and the level of at least one other
IRC marker expression product from LIST F.
287. A method according to claim 284, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E and the level of at least one other
IRC marker expression product from LIST F.
288. A method according to claim 284, comprising detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST C and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E and the level of at least one other
IRC marker expression product from LIST F.
289. A method according to claim 284, comprising detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST B and the level of at least one other IRC marker expression
product from LIST D and the level of at least one other IRC marker
expression product from LIST E and the level of at least one other
IRC marker expression product from LIST F.
290. A method according to any one of claims 1 to 245, comprising
detecting the level of at least one IRC marker expression product
from each of LISTS A, B, C, D, E and F.
291. A method according to claim 5, comprising diagnosing the
absence of sepsis, inSIRS or post surgical inflammation when the
measured level or functional activity of the or each IRC expression
product is the same as or similar to the measured level or
functional activity of the or each corresponding expression product
when the control subject is a normal subject.
292. A method according to claim 291, wherein the measured level of
an individual IRC expression product varies from the measured level
of an individual corresponding expression product by no more than
about 20%.
293. A method according to claim 5, wherein the biological sample
comprises blood, especially peripheral blood, which suitably
includes leukocytes.
294. A method for treating, preventing or inhibiting the
development of at least one condition selected from sepsis, inSIRS
or post-surgical inflammation in a subject, the method comprising:
comparing the level of at least one IRC expression product of a
multi-transcript-producing gene in the subject to the level of a
corresponding IRC marker expression product in at least one control
subject selected from: a post-surgical inflammation-positive
subject, an inSIRS positive subject, and a sepsis-positive subject,
wherein a difference between the level of the at least one IRC
marker expression product and the level of the corresponding IRC
marker expression product indicates whether the subject has, or is
at risk of developing, one of the conditions, wherein the at least
one IRC marker expression product is predetermined as being
differentially expressed between at least two of the conditions and
wherein at least one other expression product from the
multi-transcript producing gene is predetermined as being not so
differentially expressed; and administering to the subject, on the
basis that the subject tests positive for sepsis, an effective
amount of an agent that treats or ameliorates the symptoms or
reverses or inhibits the development of sepsis, or administering to
the subject, on the basis that the subject tests positive for
inSIRS, an effective amount of an agent that treats or ameliorates
the symptoms or reverses or inhibits the development of inSIRS; or
administering to the subject, on the basis that the subject tests
positive for post-surgical inflammation, an effective amount of an
agent that treats or ameliorates the symptoms or reverses or
inhibits the development of post-surgical inflammation.
295. A method according to claim 294, wherein the sepsis treatment
or agent is selected from antibiotics, intravenous fluids,
vasoactives, palliative support for damaged or distressed organs
and close monitoring of vital organs.
296. A method according to claim 294, wherein the inSIRS treatment
or agent is selected from antibiotics, steroids, intravenous
fluids, glucocorticoids, vasoactives, palliative support for
damaged or distressed organs (e.g. oxygen for respiratory distress,
fluids for hypovolemia) and close monitoring of vital organs.
297. A method according to claim 294, wherein the post-surgical
inflammation treatment or agent is selected from antibiotics,
intravenous fluids and anti-inflammatory agents.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to methods and apparatus
for diagnosis, detection of host response, monitoring, treatment or
management of sepsis, infection-negative systemic inflammatory
response syndrome (SIRS) and post-surgical inflammation in mammals.
More particularly, the present invention relates to marker genes
and their splice variant transcripts as well as their expression
products that are useful for distinguishing between sepsis and
infection-negative SIRS, including post-surgical inflammation, and
to the use of these markers in grading, monitoring, treatment and
management of these conditions. The invention has practical use in
early diagnosis, diagnosis of mild or sub-clinical sepsis or
infection-negative SIRS or post-surgical inflammation, in the
detection of specific cell immune responses as part of active or
progressive disease, in monitoring clinically affected subjects,
and in enabling better treatment and management decisions to be
made in clinically and sub-clinically affected subjects.
Additionally, the invention has practical use in monitoring and
grading patients in critical care or intensive care units for
sepsis or infection-negative SIRS or post-surgical inflammation,
and in predicting clinical outcome.
BACKGROUND OF THE INVENTION
[0002] Systemic Inflammatory Response Syndrome (SIRS) is
characterized by fever or hypothermia, leukocytosis or leukopenia,
tachypnea and tachycardia. SIRS may have an infectious or
non-infectious etiology and is described in association with
critical conditions that include pancreatitis, ischemia,
multi-trauma and severe tissue injury. Major open surgery is a
controlled form of physical insult that results in varying degrees
of systemic inflammation. In fact, it has been reported that the
occurrence of SIRS following cardiac bypass surgery (Chello et al.,
2006, Critical Care Medicine 34(3):660-667), open abdominal aortic
repair (Brown et al., 2003, Journal of Vascular Surgery
37(3):600-606) and open colorectal resection (Haga et al., 1997,
Critical Care Medicine 25(12):1994-2000) is very common, as well as
a major cause of postoperative complications including death.
Published findings by Michalopoulos and colleagues indicate 100% of
cardiac surgical patients (n=2615; mean age 60.8.7 yrs) in their
unit develop SIRS during the first 12 hours of post-operative care
(Michalopoulos et al., 2005, Intensive Care Medicine 22(1):S134).
Recent research has suggested that because of the amount of
cellular damage (necrosis) from major physical injury and trauma,
mitochondrial proteins are released into circulation and stimulate
damage-associated molecular patterns (DAMPs). This is significant
as mitochondria are cellular organelles which were originally
derived from bacteria via a process known as evolutionary
endosymbiosis. It is these DAMPS that stimulate an acute phase
response by the innate immune system that is biologically
concordant with pathogen-associated molecular patterns (PAMPs)
released during infection (Zhang et al., 2010, Nature
464:104-107).
[0003] If infection is suspected in addition to the any of the
above SIRS clinical presentations, the term sepsis is applied.
Sepsis can be defined as a systemic inflammatory response to
infection; typically a Gram negative or Gram positive bacterial or
fungal infection. However, microbiological evidence of a
circulating pathogen is not necessary to confirm the diagnosis of
sepsis. Severe sepsis includes hypotension and evidence of organ
dysfunction. When hypotension cannot be managed with intravenous
fluids, the diagnosis of septic shock is applied (Bone et al.,
1992, Chest 101:1644-55; American College of Chest
Physicians/Society of Critical Care Medicine Consensus Conference.
Definitions of sepsis and organ failure and guidelines for the use
of innovative therapies in sepsis. 1992, Crit Care Med.
20(6):864-874; Bernard et al., (PROWESS Study Group), 2001, N Engl
J Med. 344(10):699-709). It was thus recommended at the 1991
Consensus Conference that, when patients are identified as having
SIRS or multiple organ dysfunction syndrome (MODS), sequential
(i.e., daily or more frequently) risk stratification or probability
estimate techniques should be applied to describe the course of the
syndrome (Bone et al., 1992, supra; American College of Chest
Physicians/Society of Critical Care Medicine Consensus Conference,
1992, supra).
[0004] Sepsis is a life-threatening disorder and the leading cause
of mortality in the adult intensive care unit (ICU) ranging from
between 18-50% (Sundararajan et al., 2005, Crit. Care Med.
33:71-80; Finfer et al., 2004, Care Med. 30:589-596; Martin et al.,
2003, N Engl J. Med. 348:1546-1554; Australian Institute of Health
& Welfare, Canberra (2006). Mortality over the twentieth
century in Australia. Trends and patterns in major causes of death.
Mortality Surveillance Series, Number 4, p49). In developed
countries, the incidence of sepsis is expected to rise due to aging
populations, immune-compromised patients (e.g., patients on
chemotherapy, or have had a transplant or are on chronic
corticosteroids), increasing longevity of patients with chronic
diseases, antimicrobial resistance, especially in younger people,
as well as viral illnesses such as AIDS.
[0005] Antimicrobial resistance is becoming a significant problem
in critical care patient management, particularly with
Gram-negative bacilli (Hotchkiss and Donaldson. 2006, Nature
Reviews Immunology 6:813-822; Eber et al., 2010, Arch Intern Med.
170(4):374-353). Recent evidence suggests that indiscriminate use
of antibiotics has contributed to resistance and hence, guidance on
antibiotic treatment duration is now imperative in order to reduce
consumption in tertiary care ICU settings (Hanberger et al., 1999,
JAMA. 281:61-71).
[0006] Approximately 20 million cases of severe sepsis arise
globally per annum, and account for up to 135,000 deaths in Europe
and 215,000 in the USA (Neuhauser et al., 2003, JAMA.
289:885-888).
[0007] While half of these infections are estimated to be
community-acquired in the United States, research suggests that the
other half relate to hospital acquired infections (HAI), which
account for increased hospital in-patient admission by as much as
14 days, at an average cost of $46,000 per patient (Goldmann et
al., 1996, JAMA. 275:234-40). Bacterial and fungal sepsis is a
significant medical challenge not only in critical care but also
for hematology, transplant, medical oncology and post-surgical
in-patients.
[0008] Sepsis initiates a complex immunologic response that varies
over time and is dependent on pre-existing co-morbidities. Although
recent research demonstrates that both inflammatory and
anti-inflammatory responses are occurring in this condition, during
the early host response to microbial invasion, there is generally a
hyperinflammatory signal. That is, the majority of the sepsis cases
are the product of bacteria and fungi that do not ordinarily cause
systemic disease in immunocompetent hosts. The local innate immune
mechanisms essentially stimulate the release of cytokines,
chemokines, prostanoids and leukotrienes that increase blood flow
to local sources of infection and result in an influx of white
blood cells. During this processes, toll-like receptors (TLRs) are
also activated as part of the innate immune response and have
direct anti-microbial activity in addition to influencing the
antigen-specific adaptive response. TLRs are a type of pattern
recognition receptor that can identify PAMPs as soon as microbes
breach dermal or intestinal barriers (Hotchkiss et al., 2009,
Nature Medicine. 15(5):496-497). However, weaknesses in the innate
host defence and release of endotoxins or other virulence factors
can quickly lead to severe sepsis following a strong inflammatory
response.
[0009] For many decades, the cornerstone of sepsis diagnosis and
treatment has been identifying the causative circulating pathogen
and quantitating single immune-related blood analytes--medical
determinants which are not necessarily specific to sepsis, but
routinely conducted to assess the patient's physiological response
to the pathogen. Currently, the gold standard for detection of
bacteria and fungi is blood culture in microbiological media with
the aim of growing the causative organism. This method typically
requires between 48-72 hours of incubation before the microbe can
be identified and antibiotic sensitivity provided, such that
evidence-based treatment can be implemented in comparison to the
initial empiric practices. In contrast, it has recently been
proposed by Hotchkiss et al. (Adib-Conquy et al, 2009, Thromb
Haemast. 101(1):36-47) that the development of sepsis represents
the harmful consequences of an exuberant innate immune response.
While most patients survive this "hyperinflammatory phase," it was
suggested that what follows is a stage of protracted
immunosuppression that is referred to as immunoparalysis (Monneret
et al, 2008, Mol Med. 14(1-2):64-78; Wade et al, 2009, Science
326:865-876). This secondary immunosuppression has been
characterized by the loss of delayed type hypersensitivity response
to positive control antigens, failure to clear the primary
infection and development of secondary infections which can include
activation of normally latent viruses such as CMV or HHV. Taken
together, this implies that current clinical focus should be on
enhancing/maintaining immune competence in critically ill patients.
Thus, to achieve such a clinical goal there needs to be a method of
monitoring the status of the immune system so that immunotherapy
can be timed appropriately.
[0010] In terms of treatment and management plans; SIRS (also
referred to herein as "infection-negative SIRS") and sepsis are
quite different. On initial presentation to the Emergency
Department, a patient displaying two or more SIRS criteria will be
treated with intravenous glucocorticosteroids (GCS) and
antibiotics, even if infection is only suspected. Empiric treatment
will continue until positive microbiology findings are known, past
medical history is confirmed and/or there has been a positive
clinical response to early management. If it is clear, based on
clinical presentation and reason for admission, that the SIRS
response is related to acute trauma, for example motor vehicle
injury or an acute inflammatory condition such as anaphylaxis, the
patient will be managed with other intravenous fluids, blood
products or adrenaline, where indicated. However, it is important
that a patient with a true SIRS response is definitively managed as
early as possible so to conserve antibiotic efficacy. Likewise, it
is essential that a patient with a bacterial or fungal infection be
managed with antibiotics and not steroids so that immune function
is not compromised. Differential diagnosis is exponentially more
difficult when a patient presents to the Emergency Department with
clinically significant changes to vital signs such as heart rate
and blood pressure in addition to a fever. These are signs and
symptoms of the early stages of infection-negative SIRS and
infection-positive SIRS (sepsis), and impossible to delineate the
two conditions clinically. However, although the two conditions can
be separated based on physiological endpoints, the molecular
biology is considered only capable of identifying changes in the
chemical signatures that appear when a severe infection is
developing.
[0011] At the present time, diagnostic practices in clinical
pathology are moving toward gene-protein-metabolite targeted
pathways, as novel molecular profiles offer the opportunity to
assess discrete yet unique changes in multiple biomarkers in a
matter of hours, and potentially minutes. The combination of high
specificity and sensitivity, low contamination risk and blood
collection, as well as processing speed has made molecular
techniques, such as quantitative real time PCR (qRT PCR)
technology, an efficient alternative in comparison to
microbiological culture.
[0012] Given that the majority of patients (>80%) admitted to
the tertiary care ICU setting have SIRS of varying etiologies,
including following major surgery, it is of enormous clinical
importance that those patients who have a suspected infection or
are at high risk of infection can be identified early and be graded
and monitored, in order to initiate evidence-based and
goal-orientated medical therapy. This is critical, as the acute
management plans for SIRS with and without infection are very
different. Dependence on empiric treatment means that some patients
may be receiving excessive antibiotics while others are receiving
treatment (e.g. corticosteroids) that is immuno-suppressive because
a clear site of infection has not been identified. Furthermore,
once patients are identified as having sepsis, regular monitoring
of the immune system is considered essential for clinicians to
modulate therapy dependent on immune system status, the type of
infection and multi-organ complications that may be associated with
sepsis.
SUMMARY OF THE INVENTION
[0013] The present invention arises from the unexpected discovery
that the range of transcripts expressed from certain individual
genes in peripheral blood varies between patients with sepsis,
patients with infection-negative SIRS (also referred to herein as
"inSIRS") and patients following major surgery. In particular, the
present inventors have found that certain exons of individual genes
are differentially expressed in peripheral blood between these
conditions (also referred to herein as "condition-separating
exons") whilst others from the same genes are not so differential
expressed. Based on this discovery, the present inventors have
developed various methods and kits, which take advantage of
condition-separating exons to detect the presence, absence or risk
of development of sepsis, inSIRS and systemic inflammation
following major surgery. In certain embodiments, these assays and
kits represent a significant advance over prior art assays and kits
which have not been able to distinguish between systemic
inflammation following major surgery and infection-negative SIRS.
Accordingly, in these embodiments, the present invention provides a
means to separate these two groups from themselves and from sepsis
allowing for qualitative or quantitative grading of inflammatory
response as if there were a "continuum" of severity of inflammatory
response from post-surgical inflammation through to sepsis.
[0014] The present invention thus represents a significant advance
over current technologies for the management of sepsis, infection
negative SIRS and post-surgical inflammation. In certain
advantageous embodiments, it relies upon measuring the level of
certain markers in cells, especially circulating leukocytes, of the
host. In some embodiments where circulating leukocytes are the
subject of analysis, it is proposed that detection of the presence
or absence of a host response to sepsis and its sequelae (also
referred to herein as "sepsis-related conditions") will be feasible
at very early stages of its progression before extensive tissue
damage has occurred.
[0015] The present invention addresses the problem of
distinguishing between sepsis, infection-negative SIRS and
post-surgical inflammation by detecting a host response that may be
measured in host cells. Advantageous embodiments involve monitoring
the expression of particular gene transcripts in peripheral
leukocytes of the immune system, which may be reflected in changing
patterns of RNA levels or protein production that correlate with
the presence of active disease or response to disease.
[0016] Accordingly, in one aspect, the present invention provides
methods for assessing whether a subject has, or is at risk of
developing, one of a plurality of conditions selected from sepsis,
infection-negative SIRS (hereafter referred to as "inSIRS") and
post-surgical inflammation. These methods generally comprise
comparing the level of at least one expression product (also
referred to herein as an "inflammatory response continuum" (IRC)
marker expression product") of a multi-transcript-producing gene in
the subject to the level of a corresponding IRC marker expression
product in at least one control subject selected from: a
post-surgical inflammation-positive subject, an inSIRS positive
subject, a sepsis-positive subject and a normal subject, wherein a
difference between the level of the at least one IRC marker
expression product and the level of the corresponding IRC marker
expression product indicates whether the subject has, or is at risk
of developing, one of the conditions, wherein the at least one IRC
marker expression product is predetermined as being differentially
expressed between at least two of the conditions and wherein at
least one other expression product from the multi-transcript
producing gene is predetermined as being not so differentially
expressed. The at least one ICR marker expression product is
suitably selected from an ICR marker transcript or an ICR marker
polypeptide.
[0017] In some embodiments, the multi-transcript-producing gene is
selected from the group consisting of: ankyrin repeat and death
domain containing 1A (ANKDD1A) gene, rho 2 (GABRR2) gene,
orthodenticle homeobox 1 (OTX1) gene, pannexin 2 (PANX2) gene,
rhomboid 5 homolog 2 (Drosophila) (RHBDF2) gene, SLAM family member
7 (SLAMF7) gene, autophagy/beclin-1 regulator 1 (AMBRA1) gene,
carboxylesterase 2 (intestine, liver) (CES2) gene, caseinolytic
peptidase B homolog (E. coli) (CLPB) gene, homeodomain interacting
protein kinase 2 (HIPK2) gene and chromosome 1 open reading frame
91 (C1ORF91) gene, N-deacetylase/N-sulfotransferase (heparan
glucosaminyl) 1 (NDST1) gene, solute carrier family 36
(proton/amino acid symporter) (member 1 (SLC36A1) gene, ADAM
metallopeptidase domain 19 (meltrin beta) (ADAM19) gene, cullin 7
(CUL7) gene, thyroglobulin (TG) gene, programmed cell death 1
ligand 2 (PDCD1LG2) gene, glutamate receptor (ionotropic (N-methyl
D-aspartate-like 1A (GRINL1A) gene, mahogunin (ring finger 1
(MGRN1) gene, syntrophin (beta 2 (dystrophin-associated protein A 1
(59 kDa (basic component 2) (SNTB2) gene, cyclin-dependent kinase 5
(regulatory subunit 1 (p35) (CDK5R1) gene, glucosidase (alpha; acid
(GAA) gene, katanin p60 subunit A-like 2 (KATNAL2) gene,
carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4)
gene, zinc finger protein 335 (ZNF335) gene, aspartate
beta-hydroxylase domain containing 2 (ASPHD2) gene, acidic repeat
containing (ACRC) gene, butyrophilin-like 3/butyrophilin-like 8
(BTNL3, BTLN8) gene, Moloney leukemia virus 10 homolog (mouse)
(MOVE)) gene, mediator complex subunit 12-like (MED12L) gene,
kelch-like 6 (Drosophila) (KLHL6) gene, PDZ and LIM domain 5
(PDLIM5) gene, UDP-N-acetyl-alpha-D-galactosamine:polypeptide
N-acetylgalactosaminyltransferase 10 (GALNT10) gene, secernin 1
(SCRN1) gene, vesicular (overexpressed in cancer (prosurvival
protein 1 (VOPP1, RP11-289I10.2) gene, FK506 binding protein 9, 63
kDa (FKBP9, FKBP9, FKBP9L, AC091812.2) gene, kinesin family member
27 (KIF27) gene, piwi-like 4 (Drosophila) (PIWIL4) gene,
telomerase-associated protein 1 (TEP1) gene, GTP cyclohydrolase 1,
(GCH1) gene, proline rich 11, (PRR11) gene, cadherin 2, type 1,
N-cadherin (neuronal) (CDH2) gene, protein phosphatase 1B-like
(FLJ40125, AC138534.1) (PPMIN) gene, related RAS viral (r-ras)
oncogene homolog, (RRAS) gene,
dolichyl-diphosphooligosaccharide-protein glycosyltransferase,
(DDOST) gene, anterior pharynx defective 1 homolog A (C. elegans)
(APH1A) gene, tubulin tyrosine ligase (TTL) gene, testis expressed
261, (TEX261) gene, coenzyme Q2 homolog, prenyltransferase (yeast)
(COQ2) gene, FCH and double SH3 domains 1, (FCHSD1) gene,
BCL2-antagonist/killer 1, (BAK1) gene, solute carrier family 25
(mitochondrial carrier; phosphate carrier) member 25, (SLC25A25)
gene, RELT tumor necrosis factor receptor, (RELT) gene, acid
phosphatase 2, lysosomal, (ACP2) gene, TBC1 domain family, member
2B, (TBC1D2B) gene, Fanconi anemia, complementation group A,
(FANCA) gene, solute carrier family 39 (metal ion transporter)
member 11, (SLC39A11) gene.
[0018] In some embodiments, the methods comprise comparing the
level of at least one IRC marker transcript to the level of a
corresponding IRC marker transcript, wherein the IRC marker
transcript is selected from the group consisting of: (a) a
polynucleotide comprising a nucleotide sequence that shares at
least 70% (or at least 71% to at least 99% and all integer
percentages in between) sequence identity with the sequence set
forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,
55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,
89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,
247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,
273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,
325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,
351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,
377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401,
403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427,
429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453,
455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,
481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505,
507, 509, 511, 513 or 515, or a complement thereof; (b) a
polynucleotide comprising a nucleotide sequence that encodes a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98; 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,
176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,
202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,
306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,
332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,
358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,
410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,
436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,
462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,
488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,
514 or 516; (c) a polynucleotide comprising a nucleotide sequence
that encodes a polypeptide that shares at least 70% (or at least
71% to at least 99% and all integer percentages in between)
sequence similarity or identity with at least a portion of the
sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,
90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,
118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,
144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,
170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194,
196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220,
222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246,
248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272,
274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298,
300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324,
326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350,
352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376,
378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402,
404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428,
430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,
456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480,
482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506,
508, 510, 512, 514 or 516; (d) a polynucleotide expression product
comprising a nucleotide sequence that hybridizes to the sequence of
(a), (b), (c) or a complement thereof, under at least medium or
high stringency conditions.
[0019] In some embodiments, the methods comprise comparing the
level of at least one IRC marker polypeptide to the level of a
corresponding IRC marker polypeptide, wherein the IRC marker
polypeptide is selected from the group consisting of: (i) a
polypeptide comprising the amino acid sequence set forth in any one
of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,
98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174,
176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,
202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278,
280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304,
306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330,
332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356,
358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382,
384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408,
410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434,
436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460,
462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486,
488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512,
514 or 516; and (ii) a polypeptide comprising an amino acid
sequence that shares at least 70% (or at least 71% to at least 99%
and all integer percentages in between) sequence similarity or
identity with the sequence set forth in any one of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260;
262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,
314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,
340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,
366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,
444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,
470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514 or 516.
[0020] In some embodiments, the methods comprise: (1) measuring in
a biological sample obtained from the subject the level of the at
least one IRC marker expression product and (2) comparing the
measured level of each IRC marker expression product to the level
of a corresponding IRC marker expression product in a reference
sample obtained from the at least one control subject. In
illustrative examples of this type, the methods comprise assessing
whether the subject has, or is at risk of developing, one of the
plurality of conditions when the measured level of the or each IRC
marker expression product is different than the measured level of
the or each corresponding IRC marker expression product. In
specific embodiments, the level of an individual IRC marker
expression product is at least 110%, 120%, 130%, 140%, 150%, 160%,
170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or
1000%, or no more than about 95%, 90%, 80%, 70%, 60%, 50%, 40%,
30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or
0.0001% of the level of an individual corresponding IRC expression
product, which is hereafter referred to as "differential
expression."
[0021] In some embodiments, the presence or risk of development of
sepsis is determined by detecting in the subject a decrease in the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or
48 IRC marker expression products from a multi-transcript-producing
gene selected from the group consisting of: KIF27, OTX1, CDK5R1,
FKBP9, CDH2, ADAM19, BTNL3/8 and PANX2 (hereafter referred to as
"LIST A"), as compared to the level of a corresponding IRC marker
expression product(s) in a post-surgical inflammation-positive
control subject. In some embodiments, the presence or risk of
development of post-surgical inflammation is determined by
detecting in the subject an increase in the level of at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 IRC marker expression
product(s) from at least one multi-transcript-producing gene
selected from the group consisting of KIF27, OTX1, CDK5R1, FKBP9,
CDH2, ADAM19, BTNL3/8 and PANX2(i.e., LIST A), as compared to the
level of a corresponding IRC marker expression product in a sepsis
control subject. In illustrative examples of these embodiments, the
KIF27 IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from KIF27 exon 4 and exon 7, or
an amino acid sequence encoded by that exon. Representative KIF27
IRC transcripts are set forth in SEQ ID NO: 1, 3, 5, 7 and 9 and
representative KIF27 IRC polypeptides are set forth in SEQ ID NO:
2, 4, 6, 8, and 10. In other illustrative examples, the OTX1 IRC
marker expression product comprises a nucleotide sequence
corresponding to OTX1 exon 5 or an amino acid sequence encoded by
that exon. Representative OTX1 IRC transcripts are set forth in SEQ
ID NO: 11 and 13 and representative OTX1 IRC polypeptides are set
forth in SEQ ID NO: 12 and 14. In still other illustrative
examples, the CDK5R1 IRC marker expression product comprises a
nucleotide sequence corresponding to CDK5R1 exon 2, or an amino
acid sequence encoded by that exon. A representative CDK5R1 IRC
transcript is set forth in SEQ ID NO: 15 and a representative
CDK5R1 IRC polypeptide is set forth in SEQ ID NO: 16. In still
other illustrative examples, the FKBP9 IRC marker expression
product comprises a nucleotide sequence corresponding to FKBP9 exon
10, or amino acid sequence(s) encoded by that exon. A
representative FKBP9 IRC transcript is set forth in SEQ ID NO: 17
and a representative FKBP9 IRC polypeptide is set forth in SEQ ID
NO: 18. In still other illustrative examples, the CDH2 IRC marker
expression product comprises a nucleotide sequence corresponding to
CDH2 exon 10, or an amino acid sequence encoded by that exon.
Representative CDH2 IRC transcripts are set forth in SEQ ID NO: 19
and 21, and representative CDH2 IRC polypeptides are set forth in
SEQ ID NO: 20 and 22. In still other illustrative examples, the
ADAM19 IRC marker expression product comprises a nucleotide
sequence corresponding to ADAM19 exon 10, or an amino acid sequence
encoded by that exon. Representative ADAM19 IRC transcripts are set
forth in SEQ ID NO: 23, 25, 27 and 29, and representative ADAM19
IRC polypeptides are set forth in SEQ ID NO: 24, 26, 28 and 30. In
still other illustrative examples, the BTNL8/3 IRC marker
expression product comprises a nucleotide sequence corresponding to
BTNL8/3 exon 6, or an amino acid sequence encoded by that exon.
Representative BTNL8/3 IRC transcripts are set forth in SEQ ID NO:
31, 33, 35, 37, 39 and 41, and representative BTNL8/3 IRC
polypeptides are set forth in SEQ ID NO: 32, 34, 36, 38, 40 and 42.
In other illustrative examples, the PANX2 IRC marker expression
product comprises a nucleotide sequence corresponding to an exon
selected from PANX2 exon 1 and exon 2, or an amino acid sequence
encoded by that exon. Illustrative PANX2 IRC transcripts are set
forth in SEQ ID NO: 43, 45 and 47 and illustrative PANX2 IRC
polypeptides are set forth in SEQ ID NO: 44, 46 and 48. Information
on each gene in LIST A exhibiting splice variation and ability to
determine the presence or risk of sepsis versus post-surgical
inflammation, the corresponding sequence numbers, log fold changes
(and direction), T adjusted P value, relevant exon number and
number of possible exons in the gene, is presented in Table 7.
[0022] In some embodiments, the presence or risk of development of
sepsis is determined by detecting in the subject an increase in the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155, 156, 157 or 158 IRC marker expression
product(s) from at least one multi-transcript-producing gene
selected from the group consisting of: PDLIM5, SCRN1, ASPHD2,
VOPP1, ACRC, GALNT10, AC1385341, MED12L, RHBDF2, KLHL6, TEP1,
PIWIL6, PRR1, RRAS, TG, ANKDD1A, GABRR2, MOV10, SLAMF7, PDCDILG2
and GCH1 (hereafter referred to as "LIST B"), as compared to the
level of a corresponding IRC marker expression product in a
post-surgical-positive subject control subject. In some
embodiments, the presence or risk of development of post-surgical
inflammation is determined by detecting in the subject a decrease
in the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,
65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,
82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,
125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,
138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,
151, 152, 153, 154, 155, 156, 157 or 158 IRC marker expression
product(s) from at least one multi-transcript-producing gene
selected from the group consisting of: PDLIM5, SCRN1, ASPHD2,
VOPP1, ACRC, GALNT10, AC1385341, MED12L, RHBDF2, KLHL6, TEP1,
PIWIL6, PRR1, RRAS, TG, ANKDD1A, GABRR2, MOV10, SLAMF7, PDCDILG2
and GCH1 (i.e., LIST B), as compared to the level of a
corresponding IRC marker expression product in a sepsis control
subject. In illustrative examples of these embodiments, the PDLIM5
IRC marker expression product comprises a nucleotide sequence
corresponding to PDLIM5 exon 5 or an amino acid sequence encoded by
that exon. A non-limiting PDLIM5 IRC transcript is set forth in SEQ
ID NO: 49 and a non-limiting PDLIM5 IRC polypeptide is set forth in
SEQ ID NO: 50. In still other illustrative examples, the SCRN1 IRC
marker expression product comprises a nucleotide sequence
corresponding to SCRN1 exon 5 or an amino acid sequence encoded by
that exon. Representative SCRN1 IRC transcripts are set forth in
SEQ ID NO: 51, 53, 55, 57, 59, 61 and 63, and representative SCRN1
IRC polypeptides are set forth in SEQ ID NO: 52, 54, 56, 58, 60, 62
and 64. In still other illustrative examples, the ASPHD2 IRC marker
expression product comprises a nucleotide sequence corresponding to
ASPHD2 exon 4 or an amino acid sequence encoded by that exon.
Representative ASPHD2 IRC transcripts are set forth in SEQ ID NO:
65, 67 and 69, and representative ASPHD2 IRC polypeptides are set
forth in SEQ ID NO: 66, 68 and 70. In still other illustrative
examples, the VOPP1 IRC marker expression product comprises a
nucleotide sequence corresponding to VOPP1 exon 3 or an amino acid
sequence encoded by that exon. Representative VOPP1 IRC transcripts
are set forth in SEQ ID NO: 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,
91 and 93, and representative VOPP1 IRC polypeptides are set forth
in SEQ ID NO: 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92 and 94. In
still other illustrative examples, the ACRC IRC marker expression
product comprises a nucleotide sequence corresponding to one or
both exons selected from ACRC exons 3 and 5, or amino acid
sequence(s) encoded by one or both of those exons. Non-limiting
ACRC IRC transcripts are set forth in SEQ ID NO: 95 and 97, and
non-limiting ACRC IRC polypeptides are set forth in SEQ ID NO: 96
and 98. In still other illustrative examples, the GALNT10 IRC
marker expression product comprises a nucleotide sequence
corresponding to GALNT10 exon 6 or an amino acid sequence encoded
by that exon. Representative GALNT10 IRC transcripts are set forth
in SEQ ID NO: 99 and 101, and representative GALNT10 IRC
polypeptides are set forth in SEQ ID NO: 100 and 102. In still
other illustrative examples, the AC1385341 IRC marker expression
product comprises a nucleotide sequence corresponding to AC1385341
exon 3 or an amino acid sequence encoded by that exon.
Representative AC1385341 IRC transcripts are set forth in SEQ ID
NO: 103, 105, 107, 109, 111, 113, 115, 117, 119, 121 and 123, and
representative AC1385341 IRC polypeptides are set forth in SEQ ID
NO: 104, 106, 108, 110, 112, 114, 116, 118, 120, 122 and 124. In
still other illustrative examples, the MED12L IRC marker expression
product comprises a nucleotide sequence corresponding to MED12L
exon 17 or an amino acid sequence encoded by that exon.
Representative MED12L IRC transcripts are set forth in SEQ ID NO:
125 and 127, and representative MED12L IRC polypeptides are set
forth in SEQ ID NO: 126 and 128. In still other illustrative
examples, the RHBDF2 IRC marker expression product comprises a
nucleotide sequence corresponding to an exon selected from RHBDF2
exons 6, 9, 10, 11, 14, 17, 18 or 19, or an amino acid sequence
encoded by that exon. Representative RHBDF2 IRC transcripts are set
forth in SEQ ID NO: 129, 131 and 133 and representative RHBDF2 IRC
polypeptides are set forth in SEQ ID NO: 130, 132 and 134. In still
other illustrative examples, the KLHL6 IRC marker expression
product comprises a nucleotide sequence corresponding to KLHL6 exon
7 or an amino acid sequence encoded by that exon. A representative
KLHL6 IRC transcript is set forth in SEQ ID. NO: 135, and a
representative KLHL6 IRC polypeptide is set forth in SEQ ID NO:
136. In other illustrative examples, the TEP1 IRC marker expression
product comprises a nucleotide sequence corresponding to TEP1 exon
49, or an amino acid sequence encoded by that exon. Non-limiting
TEP1 IRC transcripts are set forth in SEQ ID NO: 137 and 139, and
non-limiting TEP1 IRC polypeptides are set forth in SEQ ID NO: 138
and 140. In still other illustrative examples, the PIWIL6 IRC
marker expression product comprises a nucleotide sequence
corresponding to one or both exons selected from PIWIL6 exons 2 and
14, or amino acid sequence(s) encoded by one or both of those
exons. Non-limiting PIWIL6 IRC transcripts are set forth in SEQ ID
NO: 141 and 143, and non-limiting PIWIL6 IRC polypeptides are set
forth in SEQ ID NO: 142 and 144. In still other illustrative
examples, the PRR11 IRC marker expression product comprises a
nucleotide sequence corresponding to one or both exons selected
from PRR11 exons 4 and 5, or amino acid sequence(s) encoded by one
or both of those exons. A non-limiting PRR11 IRC transcript is set
forth in SEQ ID NO: 145, and a non-limiting PRR11 IRC polypeptide
is set forth in SEQ ID NO: 146. In still other illustrative
examples, the RRAS IRC marker expression product comprises a
nucleotide sequence corresponding to RRAS exon 1 or an amino acid
sequence encoded by that exon. A representative BRAS IRC transcript
is set forth in SEQ ID NO: 147, and a representative RRAS IRC
polypeptide is set forth in SEQ ID NO: 148. In other illustrative
examples, the TG IRC marker expression product comprises a
nucleotide sequence corresponding to TG exon 6, or an amino acid
sequence encoded by that exon. Non-limiting TG IRC transcripts are
set forth in SEQ ID NO: 149 and 151, and non-limiting TG IRC
polypeptides are set forth in SEQ ID NO: 150 and 152. In other
illustrative examples, the ANKDD1A IRC marker expression product
comprises a nucleotide sequence corresponding to ANKDD1A exon 7 or
an amino acid sequence encoded by that exon. Non-limiting ANKDD1A
IRC transcripts are set forth in SEQ ID NO: 153, 155, 157, 159 and
161 and non-limiting ANKDD1A IRC polypeptides are set forth in SEQ
ID NO:154, 156, 158, 160 and 162. In other illustrative examples,
the GABRR2 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from GABRR2 exons 7, 8
or 9 or an amino acid sequence encoded by that exon. Illustrative
GABRR2 IRC transcripts are set forth in SEQ ID NO: 163 and 165 and
illustrative GABRR2 IRC polypeptides are set forth in SEQ ID NO:
164 and 166. In still other illustrative examples, the MOV10 IRC
marker expression product comprises a nucleotide sequence
corresponding to MOV10 exon 6 or an amino acid sequence encoded by
that exon. Representative MOV10 IRC transcripts are set forth in
SEQ ID NO: 167, 169, 171, 173, 175 and 177, and representative
MOV10 IRC polypeptides are set forth in SEQ ID NO: 168, 170, 172,
174, 176 and 178. In still other illustrative examples, the SLAMF7
IRC marker expression product comprises a nucleotide sequence
corresponding to an exon selected from SLAMF7 exons 2, 3, 4 or 5,
or an amino acid sequence encoded by that exon. Non-limiting SLAMF7
IRC transcripts are set forth in SEQ ID NO: 179, 181, 183, 185,
187, 189, 191 and 193 and non-limiting SLAMF7 IRC polypeptides are
set forth in SEQ ID NO: 180, 182, 184, 186, 188, 190, 192, and 194.
In still other illustrative examples, the PDCILG2 IRC marker
expression product comprises a nucleotide sequence corresponding to
one or both exons selected from PDCILG2 exons 1 and 2, or amino
acid sequence(s) encoded by one or both of those exons.
Non-limiting PDCILG2 IRC transcripts are set forth in SEQ ID NO:
195 and 197, and non-limiting PDCILG2 IRC polypeptides are set
forth in SEQ ID NO: 196 and 198. In still other illustrative
examples, the GCH1 IRC marker expression product comprises a
nucleotide sequence corresponding to GCH1 exon 2 or an amino acid
sequence encoded by that exon. Representative GCH1 IRC transcripts
are set forth in SEQ ID NO: 199, 201, 203 and 205, and
representative GCH1 IRC polypeptides are set forth in SEQ ID NO:
1200, 202, 204 and 206. Information on each gene in LIST B
exhibiting splice variation and ability to determine the presence
or risk of sepsis versus post-surgical inflammation, the
corresponding sequence numbers, log fold changes (and direction), T
adjusted P value, relevant exon number and number of possible exons
in the gene, is presented in Table 7.
[0023] In some embodiments, the presence or risk of development of
sepsis is determined by detecting in the subject an increase in the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,
83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,
100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,
113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,
126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,
139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151,
152, 153, 154, 155 or 156 IRC marker expression product(s) from at
least one multi-transcript-producing gene selected from the group
consisting of: RELT, ACP2, FCHSD1, CLPB, SLC39A 1, TBC1D2B, APH1A,
DDOST, BAK1, SLC25A25A, COQ2, FANCA, PIWIL4, ZNF335, TEX261,
GABRR2, VOPP1, TTL, CES2, GALNT10, C1ORF91, AMBRA1 and SCRN1
(hereafter referred to as "LIST C"), as compared to the level of a
corresponding IRC marker expression product in an inSIRS-positive
control subject. In some embodiments, the presence or risk of
development of inSIRS is determined by detecting in the subject a
decrease in the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,
110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,
123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,
136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
149, 150, 151, 152, 153, 154, 155 or 156 IRC marker expression(s)
product from at least one multi-transcript-producing gene selected
from the group consisting of: RELT, ACP2, FCHSD1, CLPB, SLC39A1,
TBC1D2B, APH1A, DDOST, BAK1, SLC25A25A, COQ2, FANCA, PIWIL4,
ZNF335, TEX261, GABRR2, VOPP1, TTL, CES2, GALNT10, C1ORF91, AMBRA1
and SCRN1 (i.e., LIST C), as compared to the level of the
corresponding IRC marker expression product in a sepsis-positive
control subject. In illustrative examples of these embodiments, the
RELT IRC marker expression product comprises a nucleotide sequence
corresponding to RELT exon 4 or an amino acid sequence encoded by
that exon. Illustrative RELT IRC transcripts are set forth in SEQ
ID NO: 307 and 209 and illustrative RELT IRC polypeptides are set
forth in SEQ ID NO: 208 and 210. In other illustrative examples,
the ACP2 IRC marker expression product comprises a nucleotide
sequence corresponding to ACP2 exon 7 or an amino acid sequence
encoded by that exon. A non-limiting ACP2 IRC transcript is set
forth in SEQ ID NO: 211 and a non-limiting ACP2 IRC polypeptide is
set forth in SEQ ID NO: 212. In still other illustrative examples,
the FCHSD1 IRC marker expression product comprises a nucleotide
sequence corresponding to FCHSD1 exon 14 or an amino acid sequence
encoded by that exon. Illustrative FCHSD1 IRC transcripts are set
forth in SEQ ID NO: 213 and 215 and illustrative FCHSD1 IRC
polypeptides are set forth in SEQ ID NO: 214 and 216. In still
other illustrative examples, the CLPB IRC marker expression product
comprises a nucleotide sequence corresponding to CLPB exon 10 or an
amino acid sequence encoded by that exon. Representative CLPB IRC
transcripts are set forth in SEQ ID NO: 217, 219 and 221 and
representative CLPB IRC polypeptides are set forth in SEQ ID NO:
218, 220 and 222. In other illustrative examples, the SLC39A11 IRC
marker expression product comprises a nucleotide sequence
corresponding to SLC39A11 exon 2 or an amino acid sequence encoded
by that exon. A non-limiting SLC39A11 IRC transcript is set forth
in SEQ ID NO: 223 and a non-limiting SLC39A11 IRC polypeptide is
set forth in SEQ ID NO: 224. In other illustrative examples, the
TBC1D2B IRC marker expression product comprises a nucleotide
sequence corresponding to TBC1D2B exon 13 or an amino acid sequence
encoded by that exon. Illustrative TBC1D2B IRC transcripts are set
forth in SEQ ID NO: 225, 227 and 229 and illustrative TBC1D2B IRC
polypeptides are set forth in SEQ ID NO: 226, 228 and 230. In still
other illustrative examples, the APH1A IRC marker expression
product comprises a nucleotide sequence corresponding to APH1A exon
1 or an amino acid sequence encoded by that exon. Illustrative
APH1A IRC transcripts are set forth in SEQ ID NO: 231, 233, 235,
237, 239 and 241 and illustrative APH1A IRC polypeptides are set
forth in SEQ ID NO: 232, 234, 236, 238, 240 and 242. In other
illustrative examples, the DDOST IRC marker expression product
comprises a nucleotide sequence corresponding to DDOST exon 2 or an
amino acid sequence encoded by that exon. A non-limiting DDOST IRC
transcript is set forth in SEQ ID NO: 243 and a non-limiting DDOST
IRC polypeptide is set forth in SEQ ID NO: 244. In still other
illustrative examples, the BAK1 IRC marker expression product
comprises a nucleotide sequence corresponding to BAK1 exon 7 or an
amino acid sequence encoded by that exon. Illustrative BAK1 IRC
transcripts are set forth in SEQ ID NO: 245 and 247 and
illustrative BAK1 IRC polypeptides are set forth in SEQ ID NO: 246
and 248. In still other illustrative examples, the SLC25A25A IRC
marker expression product comprises a nucleotide sequence
corresponding to SLC25A25A exon 10 or an amino acid sequence
encoded by that exon. Illustrative SLC25A25A IRC transcripts are
set forth in SEQ ID NO: 249, 251, 253, 255, 257, 259 and 261 and
illustrative. SLC25A25A IRC polypeptides are set forth in SEQ ID
NO: 250, 252, 254, 256, 258, 260 and 262. In still other
illustrative examples, the COQ1 IRC marker expression product
comprises a nucleotide sequence corresponding to COQ1 exon 1 or an
amino acid sequence encoded by that exon. Illustrative COQ1 IRC
transcripts are set forth in SEQ ID NO: 263, 265 and 267 and
illustrative COQ1 IRC polypeptides are set forth in SEQ ID NO: 264,
266 and 268. In still other illustrative examples, the FANCA IRC
marker expression product comprises a nucleotide sequence
corresponding to FANCA exon 35 or an amino acid sequence encoded by
that exon. Illustrative FANCA IRC transcripts are set forth in SEQ
ID NO: 269 and 271 and illustrative FANCA IRC polypeptides are set
forth in SEQ ID NO: 270 and 272. In other illustrative examples,
the PIWIL4 IRC marker expression product comprises a nucleotide
sequence corresponding to one or both exons selected from PIWIL4
exons 2 and 14, or amino acid(s) sequence encoded by one or both of
those exons. Non-limiting PIWIL4 IRC transcripts are set forth in
SEQ ID NO: 273 and 275 and non-limiting PIWIL4 IRC polypeptides are
set forth in SEQ ID NO: 274 and 276. In still other illustrative
examples, the ZNF335 IRC marker expression product comprises a
nucleotide sequence corresponding to ZNF335 exon 5 or an amino acid
sequence encoded by that exon. Illustrative ZNF335 IRC transcripts
are set forth in SEQ ID NO: 277, 279 and 281 and illustrative
ZNF335 IRC polypeptides are set forth in SEQ ID NO: 278, 280 and
282. In still other illustrative examples, the TEX261 IRC marker
expression product comprises a nucleotide sequence corresponding to
TEX261 exon 3 or an amino acid sequence encoded by that exon.
Illustrative TEX261 IRC transcripts are set forth in SEQ ID NO: 283
and 285 and illustrative TEX261 IRC polypeptides are set forth in
SEQ ID NO: 284 and 286. In other illustrative examples, the GABRR2
IRC marker expression product comprises a nucleotide sequence
corresponding to 1, 2 or each of the exons selected from GABRR2
exons 7, 8 and 9, or amino acid sequence(s) encoded by 1, 2 or each
of those exons. Non-limiting GABRR2 IRC transcripts are set forth
in SEQ ID NO: 287 and 289 and non-limiting GABRR2 IRC polypeptides
are set forth in SEQ ID NO: 288 and 290. In still other
illustrative examples, the VOPP1 IRC marker expression product
comprises a nucleotide sequence corresponding to VOPP1 exon 3 or an
amino acid sequence encoded by that exon. Illustrative VOPP1 IRC
transcripts are set forth in SEQ ID NO: 291, 293, 295, 297, 299,
301, 303, 305, 307, 309, 311 and 313 and illustrative VOPP1 IRC
polypeptides are set forth in SEQ ID NO: 292, 294, 296, 298, 300,
302, 304, 306, 308, 310, 312 and 314. In other illustrative
examples, the TTL IRC marker expression product comprises a
nucleotide sequence corresponding to TTL exon 7 or an amino acid
sequence encoded by that exon. A non-limiting TTL IRC transcript is
set forth in SEQ ID NO: 315 and a non-limiting TTL IRC polypeptide
is set forth in SEQ ID NO: 316. In other illustrative examples, the
CES2 IRC marker expression product comprises a nucleotide sequence
corresponding to CES2 exon 1 or an amino acid sequence encoded by
that exon. Illustrative CES2 IRC transcripts are set forth in SEQ
ID NO: 317 and 319 and illustrative CES2 IRC polypeptides are set
forth in SEQ ID NO: 318 and 320. In still other illustrative
examples, the GALNT10 IRC marker expression product comprises a
nucleotide sequence corresponding to GALNT10 exon 6 or an amino
acid sequence encoded by that exon. Illustrative GALNT10 IRC
transcripts are set forth in SEQ ID NO: 321 and 323 and
illustrative GALNT10 IRC polypeptides are set forth in SEQ ID NO:
322 and 324. In still other illustrative examples, the C1ORF91 IRC
marker expression product comprises a nucleotide sequence
corresponding to C1ORF91 exon 2 or an amino acid sequence encoded
by that exon. Illustrative C1ORF91 IRC transcripts are set forth in
SEQ ID NO: 325, 327, 329, 331, 333 and 335 and illustrative C1ORF91
IRC polypeptides are set forth in SEQ ID NO: 326, 328, 330, 332,
334 and 336. In other illustrative examples, the AMBRA1 IRC marker
expression product comprises a nucleotide sequence corresponding to
an exon selected from AMBRA1 exons 2 and 4, or an amino acid
sequence encoded by that exon. Non-limiting AMBRA1 IRC transcripts
are set forth in SEQ ID NO: 337, 339, 341, 343, 345 and 347 and
non-limiting AMBRA1 IRC polypeptides are set forth in SEQ ID NO:
338, 340, 342, 344, 346 and 348. In still other illustrative
examples, the SCRN1 IRC marker expression product comprises a
nucleotide sequence corresponding to SCRN1 exon 5 or an amino acid
sequence encoded by that exon. Illustrative SCRN1 IRC transcripts
are set forth in SEQ ID NO: 349, 351, 353, 355, 357, 359 and 361
and illustrative SCRN1 IRC polypeptides are set forth in SEQ ID NO:
350, 352, 354, 356, 358, 360 and 362. Information on each gene in
LIST C exhibiting splice variation and ability to determine the
presence or risk of sepsis versus post-surgical inflammation, the
corresponding sequence numbers, log fold changes (and direction), T
adjusted P value, relevant exon number and number of possible exons
in the gene, is presented in Table 8.
[0024] In some embodiments, the presence or risk of development of
sepsis is determined by detecting in the subject an decrease in the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20 IRC marker expression product(s) from at
least one multi-transcript-producing gene selected from the group
consisting of: GRINL1A and KATNAL2 (hereafter referred to as "LIST
D"), as compared to the level of a corresponding IRC marker
expression product in an inSIRS-positive control subject. In some
embodiments, the presence or risk of development of inSIRS is
determined by detecting in the subject a increase in the level of
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 IRC marker expression(s) product from at least one
multi-transcript-producing gene selected from the group consisting
of: GRINL1A and KATNAL2 (i.e., LIST D), as compared to the level of
the corresponding IRC marker expression product in a
sepsis-positive control subject. In illustrative examples of these
embodiments, the GRINL1 IRC marker expression product comprises a
nucleotide sequence corresponding to an exon selected from GRINL1
exon 5, or an amino acid sequence encoded by that exon.
Non-limiting GRINL1 IRC transcripts are set forth in SEQ ID NO:
363, 365, 367, 369, 371, 373, 375 and 377 and non-limiting GRINL1
IRC polypeptides are set forth in SEQ ID NO:364, 366, 368, 370,
372, 374, 376 and 378. In other illustrative examples, the KATNAL2
IRC marker expression product comprises a nucleotide sequence
corresponding to KATNAL2 exon 3 or an amino acid sequence encoded
by that exon. Illustrative KATNAL2 IRC transcripts are set forth in
SEQ ID NO: 379 and 381 and illustrative KATNAL2 IRC polypeptides
are set forth in SEQ ID NO: 380 and 382. Information on each gene
in LIST D exhibiting splice variation and ability to determine the
presence or risk of sepsis versus post-surgical inflammation, the
corresponding sequence numbers, log fold changes (and direction), T
adjusted P value, relevant exon number and number of possible exons
in the gene, is presented in Table 8.
[0025] In some embodiments, the presence or risk of development of
inSIRS is determined by detecting in the subject an increase in the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37 or 38, IRC marker expression product(s) from
at least one multi-transcript-producing gene selected from the
group consisting of: PDCD1LG2, KATNAL2, GRINL1A, ACRC, TG, and
ASPHD2 (hereafter referred to as "LIST E"), as compared to the
level of a corresponding IRC marker expression product in a
post-surgical inflammation-positive control subject. In other
embodiments, the presence or risk of development of post-surgical
inflammation is determined by detecting in the subject a decrease
in the level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37 or 38 IRC marker expression product(s)
from at least one multi-transcript-producing gene selected from the
group consisting of: PDCD1LG2, KATNAL2, GRINL1A, ACRC, TG, and
ASPHD2 (i.e., LIST E), as compared to the level of a corresponding
IRC marker expression product in an inSIRS-positive control
subject. In illustrative examples of these embodiments, the
PDCD1LG2 IRC marker expression product comprises a nucleotide
sequence corresponding to PDCD1LG2 exon 1, 2 or an amino acid
sequence encoded by those exons. Non-limiting PDCD1LG2 IRC
transcripts are set forth in SEQ ID NO: 383 and 385 and
non-limiting PDCD1LG21 IRC polypeptides are set forth in SEQ ID NO:
384 and 386. In other illustrative examples, the KATNAL2 IRC marker
expression product comprises a nucleotide sequence corresponding to
KATNAL2 exon 3 or an amino acid sequence encoded by that exon.
Illustrative KATNAL2 IRC transcripts are set forth in SEQ ID NO:
387 and 389 and illustrative KATNAL2 IRC polypeptides are set forth
in SEQ ID NO: 388 and 390. In other illustrative examples, the
GRINL1 IRC marker expression product comprises a nucleotide
sequence corresponding to an exon selected from GRINL1 exon 5, or
an amino acid sequence encoded by that exon. Non-limiting GRINL1
IRC transcripts are set forth in SEQ ID NO: 391, 393, 395, 397,
399, 401, 403 and 405 and non-limiting GRINL1 IRC polypeptides are
set forth in SEQ ID NO:392, 394, 396, 398, 400, 402, 404 and 406. n
still other illustrative examples, the ACRC IRC marker expression
product comprises a nucleotide sequence corresponding to one or
both exons selected from ACRC exons 3 and 5, or amino acid
sequence(s) encoded by one or both of those exons. Non-limiting
ACRC IRC transcripts are set forth in SEQ ID NO: 407 and 409, and
non-limiting ACRC IRC polypeptides are set forth in SEQ ID NO: 408
and 410. In other illustrative examples, the TG IRC marker
expression product comprises a nucleotide sequence corresponding to
TG exon 6, or an amino acid sequence encoded by that exon.
Non-limiting TG IRC transcripts are set forth in SEQ ID NO: 411 and
413, and non-limiting TG IRC polypeptides are set forth in SEQ ID
NO: 412 and 414. n still other illustrative examples, the ASPHD2
IRC marker expression product comprises a nucleotide sequence
corresponding to ASPHD2 exon 4 or an amino acid sequence encoded by
that exon. Representative ASPHD2 IRC transcripts are set forth in
SEQ ID NO: 415, 417 and 419, and representative ASPHD2 IRC
polypeptides are set forth in SEQ ID NO: 416, 418 and 420.
Information on each gene in LIST E exhibiting splice variation and
ability to determine the presence or risk of sepsis versus
post-surgical inflammation, the corresponding sequence numbers, log
fold changes (and direction), T adjusted P value, relevant exon
number and number of possible exons in the gene, is presented in
Table 9.
[0026] In some embodiments, the presence or risk of development of
inSIRS is determined by detecting in the subject a decrease in the
level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,
86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 IRC marker expression
product(s) from at least one multi-transcript-producing gene
selected from the group consisting of CUL7, BTNL8/3, PANX2,
C1ORF91, ZNF335, MGRN1, GAA, CDK5R1, SNTB2, CLPB, ADAM19, SLC36A1,
FKBP9, NDST1, HIPK2 and CEACAM4 (hereafter referred to as "LIST F")
as compared to the level of the corresponding IRC marker gene(s) in
a post-surgical inflammation-positive control subject. In other
embodiments, the presence or risk of development of post-surgical
inflammation is determined by detecting in the subject an increase
in the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 or 96 IRC marker
expression product(s) from at least one multi-transcript-producing
gene selected from the group consisting of: CUL7, BTNL8/3, PANX2,
C1ORF91, ZNF335, MGRN1, GAA, CDK5R1, SNTB2, CLPB, ADAM19, SLC36A1,
FKBP9, NDST1, HIPK2 and CEACAM4 (i.e., LIST F) as compared to the
level of the corresponding IRC marker gene(s) in an inSIRS-positive
control subject. In non-limiting examples of these embodiments, the
CUL7 IRC marker expression product comprises a nucleotide sequence
corresponding to CUL7 exon 5 or an amino acid sequence encoded by
that exon. An illustrative CUL7 IRC transcript is set forth in SEQ
ID NO: 421 and an illustrative CUL7 IRC polypeptide is set forth in
SEQ ID NO: 422. In illustrative examples, the HIPK2 IRC marker
expression product comprises a nucleotide sequence corresponding to
HIPK2 exon 11 or an amino acid sequence encoded by that exon. In
still other illustrative examples, the BTNL8/3 IRC marker
expression product comprises a nucleotide sequence corresponding to
BTNL8/3 exon 6, or an amino acid sequence encoded by that exon.
Representative BTNL8/3 IRC transcripts are set forth in SEQ ID NO:
423, 425, 427, 429, 431 and 433, and representative BTNL8/3 IRC
polypeptides are set forth in SEQ ID NO: 424, 426, 428, 430, 432
and 434. In other illustrative examples, the PANX2 IRC marker
expression product comprises a nucleotide sequence corresponding to
an exon selected from PANX2 exon 1 and exon 2, or an amino acid
sequence encoded by that exon. Illustrative PANX2 IRC transcripts
are set forth in SEQ ID NO: 435, 437 and 439 and illustrative PANX2
IRC polypeptides are set forth In SEQ ID NO: 436, 438 and 440. In
still other illustrative examples, the C1 ORF91 IRC marker
expression product comprises a nucleotide sequence corresponding to
C1ORF91 exon 2 or an amino acid sequence encoded by that exon.
Illustrative C1ORF91 IRC transcripts are set forth in SEQ ID NO:
441, 443, 445, 447, 449 and 451 and illustrative C1ORF91 IRC
polypeptides are set forth in SEQ ID NO: 442, 444, 446, 448, 450
and 452. In still other illustrative examples, the ZNF335 IRC
marker expression product comprises a nucleotide sequence
corresponding to ZNF335 exon 5 or an amino acid sequence encoded by
that exon. Illustrative ZNF335 IRC transcripts are set forth in SEQ
ID NO: 453, 455 and 457 and illustrative ZNF335 IRC polypeptides
are set forth in SEQ ID NO: 454, 456 and 458. In still other
illustrative examples, the MGRN1 IRC marker expression product
comprises a nucleotide sequence corresponding to MGRN1 exon 4 or an
amino acid sequence encoded by that exon. Illustrative MGRN1 IRC
transcripts are set forth in SEQ ID NO: 459, 461 and 463 and
illustrative MGRN1 IRC polypeptides are set forth in SEQ ID NO:
460, 462 and 464. In still other illustrative examples, the GAA IRC
marker expression product comprises a nucleotide sequence
corresponding to GAA exon 3 or an amino acid sequence encoded by
that exon. Illustrative GAA IRC transcripts are set forth in SEQ ID
NO: 465, 467 and 469 and illustrative GAA IRC polypeptides are set
forth in SEQ ID NO: 466, 468 and 470. In still other illustrative
examples, the CDK5R1 IRC marker expression product comprises a
nucleotide sequence corresponding to CDK5R1 exon 2 or an amino acid
sequence encoded by that exon. An illustrative CDK5R1 IRC
transcript is set forth in SEQ ID NO: 471, and an illustrative
CDK5R1 IRC polypeptide is set forth in SEQ ID NO: 472. In still
other illustrative examples, the SNTB2 IRC marker expression
product comprises a nucleotide sequence corresponding to SNTB2 exon
4 or an amino acid sequence encoded by that exon. An illustrative
SNTB2 IRC transcript is set forth in SEQ ID NO: 473, and an
illustrative SNTB2 IRC polypeptide is set forth in SEQ ID NO: 474.
In still other illustrative examples, the CLPB IRC marker
expression product comprises a nucleotide sequence corresponding to
CLPB exon 10, or an amino acid sequence encoded by that exon.
Representative CLPB IRC transcripts are set forth in SEQ ID NO:
475, 477 and 479 and representative CLPB IRC polypeptides are set
forth in SEQ ID NO: 476, 478 and 480. In still other illustrative
examples, the ADAM19 IRC marker expression product comprises a
nucleotide sequence corresponding to ADAM19 exon 10, or an amino
acid sequence encoded by that exon. Representative ADAM19 IRC
transcripts are set forth in SEQ ID NO: 481, 483, 485 and 487, and
representative ADAM19 IRC polypeptides are set forth in SEQ ID NO:
482, 484, 486 and 488. In still other illustrative examples, the
SLC36A1 IRC marker expression product comprises a nucleotide
sequence corresponding to SLC36A1 exon 5, or an amino acid sequence
encoded by that exon. Representative SLC36A1 IRC transcripts are
set forth in SEQ ID NO: 489, 491, 493 and 495, and representative
SLC36A1 IRC polypeptides are set forth in SEQ ID NO: 490, 492, 494
and 496. In still other illustrative examples, the FKBP9 IRC marker
expression product comprises a nucleotide sequence corresponding to
FKBP9 exon 10, or amino acid sequence(s) encoded by that exon.
Representative FKBP9 IRC transcripts are set forth in SEQ ID NO:
497 and 499 and representative FKBP9 IRC polypeptides are set forth
in SEQ ID NO: 498 and 500. In other illustrative examples, the
CEACAM4 IRC marker expression product comprises a nucleotide
sequence corresponding to 1, 2 or each of the exons selected from
CEACAM4 exon 5, exon 7 and exon 23, or amino acid sequence(s)
encoded by 1, 2 each of those exons. Illustrative CEACAM4 IRC
transcripts are set forth in SEQ ID NO: 501 and 503, and
illustrative CEACAM4 IRC polypeptides are set forth in SEQ ID NO:
502 and 504. Illustrative HIPK2 IRC transcripts are set forth in
SEQ ID NO: 505, 507, 509, and 511 and illustrative HIPK2 IRC
polypeptides are set forth in SEQ ID NO: 506, 508, 510 and 512.
Information on each gene in LIST F exhibiting splice variation and
ability to determine the presence or risk of sepsis versus
post-surgical inflammation, the corresponding sequence numbers, log
fold changes (and direction), T adjusted P value, relevant exon
number and number of possible exons in the gene, is presented in
Table 9.
[0027] In some embodiments, the methods comprise measuring the
level of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 individual IRC expression
products of each of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56 or 57 multi-transcript-producing
genes (also referred to herein as "IRC multi-transcript-producing
genes"). For example, the methods may comprise measuring the level
of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 IRC marker
polynucleotides from an IRC multi-transcript-producing gene
selected from: ANKDD1A, GABRR2, OTX1, PANX2, RHBDF2, SLAMF7,
AMBRA1, CES2, CLPB, HIPK2, C1ORF91, NDST1, SLC36A1, ADAM19, CUL7,
TG, PDCD1LG2, GRINL1A, MGRN1, SNTB2, CDK5R1, GAA, KATNAL2, CEACAM4,
ZNF335, ASPHD2, ACRC, BTNL8, MOV10, MED12L, KLHL6, PDLIM5, GALNT10,
SCRN1, VOPP1, FKBP9, KIF27, PIWIL4, TEP1, GCH1, PRR11, CDH2, PPM1N,
RRAS, DDOST, APH1A, TTL, TEX261, COQ2, FCHSD1, BAK1, SLC25A25,
RELT, ACP2, TBC1D2B, FANCA or SLC39A11, either alone or in
combination with as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12
individual IRC marker polynucleotides from each of 56, 55, 54, 53,
52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,
35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 19, 18,
17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 IRC
multi-transcript-producing genes or from 1 other IRC
multi-transcript-producing gene. In other embodiments, the methods
comprise measuring the level of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
or 12 IRC marker polypeptides from an IRC
multi-transcript-producing gene as defined herein, either alone or
in combination with as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
12 individual IRC marker polypeptides expressed from each of 56,
55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39,
38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22,
21, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2
other IRC multi-transcript-producing genes or from 1 other IRC
multi-transcript-producing gene.
[0028] In illustrative examples of this type, the methods further
comprise detecting the level of at least one IRC marker expression
product from two or more of LISTS A, B, C, D, E and F. In specific
embodiments, the methods comprise detecting the level of at least
one IRC marker expression product from one of the lists and the
level of at least one different IRC marker expression product from
another of the lists. In illustrative examples of this type, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST A and the level of at least one other
IRC marker expression product from LIST B. In other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST A and the level of at least
one other IRC marker expression product from LIST C. In still other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST A and the
level of at least one other IRC marker expression product from LIST
D. In still other illustrative examples, the methods comprise
detecting the level of at least one IRC marker expression product
from LIST A and the level of at least one other IRC marker
expression product from LIST E. In still other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST A and the level of at least
one other IRC marker expression product from LIST F. In other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST B and the
level of at least one other IRC marker expression product from LIST
C. In still other illustrative examples, the methods comprise
detecting the level of at least one IRC marker expression product
from LIST B and the level of at least one other IRC marker
expression product from LIST D. In still other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST E. In still other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST B and the
level of at least one other IRC marker expression product from LIST
F. In other illustrative examples, the methods comprise detecting
the level of at least one IRC marker expression product from LIST C
and the level of at least one other IRC marker expression product
from LIST D. In still other illustrative examples, the methods
comprise detecting the level of at least one IRC marker expression
product from LIST C and the level of at least one other IRC marker
expression product from LIST E. In still other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST C and the level of at least
one other IRC marker expression product from LIST F. In still other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST D and the
level of at least one other IRC marker expression product from LIST
E. In still other illustrative examples, the methods comprise
detecting the level of at least one IRC marker expression product
from LIST D and the level of at least one other IRC marker
expression product from LIST F. In other illustrative examples, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST E and the level of at least one other
IRC marker expression product from LIST F.
[0029] In other embodiments, the methods comprise detecting the
level of at least one IRC marker expression product from each of
three lists selected from LISTS A, B, C, D, E and F. In
illustrative examples of this type, the methods comprise detecting
the level of at least one IRC marker expression product from LIST A
and the level of at least one other IRC marker expression product
from LIST B and the level of at least one other IRC marker
expression product from LIST C. In other illustrative examples, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST A and the level of at least one other
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST D. In still other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST A and the
level of at least one other IRC marker expression product from LIST
B and the level of at least one other IRC marker expression product
from LIST E. In still other illustrative examples, the methods
comprise detecting the level of at least one IRC marker expression
product from LIST A and the level of at least one other IRC marker
expression product from LIST B and the level of at least one other
IRC marker expression product from LIST F. In other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST C and the level
of at least one other IRC marker expression product from LIST D. In
still other illustrative examples, the methods comprise detecting
the level of at least one IRC marker expression product from LIST B
and the level of at least one other IRC marker expression product
from LIST C and the level of at least one other IRC marker
expression product from LIST E. In still other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST C and the level
of at least one other IRC marker expression product from LIST F. In
other illustrative examples, the methods comprise detecting the
level of at least one IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E. In still other illustrative examples, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST C and the level of at least one other
IRC marker expression product from LIST D and the level of at least
one other IRC marker expression product from LIST F. In other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST D and the
level of at least one other IRC marker expression product from LIST
E and the level of at least one other IRC marker expression product
from LIST F.
[0030] In still other embodiments, the methods comprise detecting
the level of at least one IRC marker expression product from each
of four lists selected from LISTS A, B, C, D, E and F. In
illustrative examples of this type, the methods comprise detecting
the level of at least one IRC marker expression product from LIST A
and the level of at least one other IRC marker expression product
from LIST B and the level of at least one other IRC marker
expression product from LIST C and the level of at least one other
IRC marker expression product from LIST D. In other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST A and the level of at least
one other IRC marker expression product from LIST B and the level
of at least one other IRC marker expression product from LIST C and
the level of at least one other IRC marker expression product from
LIST E. In other illustrative examples, the methods comprise
detecting the level of at least one IRC marker expression product
from LIST A and the level of at least one other IRC marker
expression product from LIST B and the level of at least one other
IRC marker expression product from LIST C and the level of at least
one other IRC marker expression product from LIST F. In other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST A and the
level of at least one other IRC marker expression product from LIST
C and the level of at least one other IRC marker expression product
from LIST D and the level of at least one other IRC marker
expression product from LIST E. In other illustrative examples, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST A and the level of at least one other
IRC marker expression product from LIST C and the level of at least
one other IRC marker expression product from LIST D and the level
of at least one other IRC marker expression product from LIST F. In
other illustrative examples, the methods comprise detecting the
level of at least one IRC marker expression product from LIST A and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E and the level of at least one other IRC marker
expression product from LIST F. In still other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST C and the level
of at least one other IRC marker expression product from LIST D and
the level of at least one other IRC marker expression product from
LIST E. In still other illustrative examples, the methods comprise
detecting the level of at least one IRC marker expression product
from LIST B and the level of at least one other IRC marker
expression product from LIST C and the level of at least one other
IRC marker expression product from LIST D and the level of at least
one other IRC marker expression product from LIST F. In other
illustrative examples, the methods comprise detecting the level of
at least one IRC marker expression product from LIST C and the
level of at least one other IRC marker expression product from LIST
D and the level of at least one other IRC marker expression product
from LIST E and the level of at least one other IRC marker
expression product from LIST F. In other illustrative examples, the
methods comprise detecting the level of at least one IRC marker
expression product from LIST A and the level of at least one other
IRC marker expression product from LIST B and the level of at least
one other IRC marker expression product from LIST D and the level
of at least one other IRC marker expression product from LIST
E.
[0031] In still other embodiments, the methods comprise detecting
the level of at least one IRC marker expression product from each
of five lists selected from LISTS A, B, C, D, E and F. In
illustrative examples of this type, the methods comprise detecting
the level of at least one IRC marker expression product from LIST A
and the level of at least one other IRC marker expression product
from LIST B and the level of at least one other IRC marker
expression product from LIST C and the level of at least one other
IRC marker expression product from LIST D and the level of at least
one other IRC marker expression product from LIST E. In other
illustrative example's, the methods comprise detecting the level of
at least one IRC marker expression product from LIST A and the
level of at least one other IRC marker expression product from LIST
B and the level of at least one other IRC marker expression product
from LIST C and the level of at least one other IRC marker
expression product from LIST D and the level of at least one other
IRC marker expression product from LIST F. In other illustrative
examples, the methods comprise detecting the level of at least one
IRC marker expression product from LIST A and the level of at least
one other IRC marker expression product from LIST C and the level
of at least one other IRC marker expression product from LIST D and
the level of at least one other IRC marker expression product from
LIST E and the level of at least one other IRC marker expression
product from LIST F. In other illustrative examples, the methods
comprise detecting the level of at least one IRC marker expression
product from LIST B and the level of at least one other IRC marker
expression product from LIST C and the level of at least one other
IRC marker expression product from LIST D and the level of at least
one other IRC marker expression product from LIST E and the level
of at least one other IRC marker expression product from LIST F. In
other illustrative examples, the methods comprise detecting the
level of at least one IRC marker expression product from LIST B and
the level of at least one other IRC marker expression product from
LIST D and the level of at least one other IRC marker expression
product from LIST E and the level of at least one other IRC marker
expression product from LIST F and the level of at least one other
IRC marker expression product from LIST A.
[0032] In still other embodiments, the methods comprise detecting
the level of at least one IRC marker expression product from each
of LISTS A, B, C, D, E and F.
[0033] In some embodiments, the methods further comprise diagnosing
the absence of sepsis, inSIRS or post surgical inflammation when
the measured level or functional activity of the or each IRC
expression product is the same as or similar to the measured level
or functional activity of the or each corresponding expression
product when the control subject is a normal subject. In these
embodiments, the measured level or functional activity of an
individual IRC expression product varies from the measured level or
functional activity of an individual corresponding expression
product by no more than about 20%, 18%, 16%, 14%, 12%, 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1%, which is hereafter referred to
as "normal expression."
[0034] In certain embodiments, a panel of IRC marker expression
products is selected to distinguish sepsis from inSIRS, sepsis from
post-surgical inflammation, sepsis from normal, inSIRS from
post-surgical inflammation, inSIRS from normal or post-surgical
from normal with at least about 70%, 80%, 85%, 90% or 95%
sensitivity, suitably in combination with at least about 70% 80%,
85%, 90% or 95% specificity. In some embodiments, both the
sensitivity and specificity are at least about 75%, 80%, 85%, 90%
or 95%.
[0035] Advantageously, the biological sample comprises blood,
especially peripheral blood, which suitably includes leukocytes.
Suitably, the expression product is selected from a RNA molecule or
a polypeptide. In some embodiments, the expression product is the
same as the corresponding expression product. In other embodiments,
the expression product is a variant (e.g., an allelic variant) of
the corresponding expression product.
[0036] In certain embodiments, the expression product or
corresponding expression product is a target RNA (e.g., mRNA) or a
DNA copy of the target RNA whose level is measured using at least
one nucleic acid probe that hybridists under at least low, medium,
or high stringency conditions to the target RNA or to the DNA copy,
wherein the nucleic acid probe comprises at least 15 contiguous
nucleotides of an IRC marker polynucleotide. In these embodiments,
the measured level or abundance of the target RNA or its DNA copy
is normalized to the level or abundance of a reference RNA or a DNA
copy of the reference RNA that is present in the same sample.
Suitably, the nucleic acid probe is immobilized on a solid or
semi-solid support. In illustrative examples of this type, the
nucleic acid probe forms part of a spatial array of nucleic acid
probes. In some embodiments, the level of nucleic acid probe that
is bound to the target RNA or to the DNA copy is measured by
hybridization (e.g., using a nucleic acid array). In other
embodiments, the level of nucleic acid probe that is bound to the
target RNA or to the DNA copy is measured by nucleic acid
amplification (e.g., using a polymerase chain reaction (PCR)). In
still other embodiments, the level of nucleic acid probe that is
bound to the target RNA or to the DNA copy is measured by nuclease
protection assay.
[0037] In other embodiments, the expression product or
corresponding expression product is a target polypeptide whose
level is measured using at least one antigen-binding molecule that
is immuno-interactive with the target polypeptide. In these
embodiments, the measured level of the target polypeptide is
normalized to the level of a reference polypeptide that is present
in the same sample. Suitably, the antigen-binding molecule is
immobilized on a solid or semi-solid support. In illustrative
examples of this type, the antigen-binding molecule forms part of a
spatial array of antigen-binding molecule. In some embodiments, the
level of antigen-binding molecule that is bound to the target
polypeptide is measured by immunoassay (e.g., using an ELISA).
[0038] In still other embodiments, the expression product or
corresponding expression product is a target polypeptide whose
level is measured using at least one substrate for the target
polypeptide with which it reacts to produce a reaction product. In
these embodiments, the measured functional activity of the target
polypeptide is normalized to the functional activity of a reference
polypeptide that is present in the same sample.
[0039] In some embodiments, a system is used to perform the
diagnostic methods as broadly described above, which suitably
comprises at least one end station coupled to a base station. The
base station is suitably caused (a) to receive subject data from
the end station via a communications network, wherein the subject
data represents parameter values corresponding to the measured or
normalized level or functional activity of at least one expression
product in the biological sample, and (b) to compare the subject
data with predetermined data representing the measured or
normalized level or functional activity of at least one
corresponding expression product in the reference sample to thereby
determine any difference in the level or functional activity of the
expression product in the biological sample as compared to the
level or functional activity of the corresponding expression
product in the reference sample. Desirably, the base station is
further caused to provide a diagnosis for the presence, absence or
degree of post-surgical inflammation, inSIRS or sepsis. In these
embodiments, the base station may be further caused to transfer an
indication of the diagnosis to the end station via the
communications network.
[0040] In another aspect, the invention contemplates use of the
methods broadly described above in monitoring, treating or managing
post-surgical inflammation or conditions that can lead to sepsis or
inSIRS, illustrative examples of which include retained placenta,
meningitis, endometriosis, shock, toxic shock (i.e., a sequelae to
tampon use), gastroenteritis, appendicitis, ulcerative colitis,
Crohn's disease, inflammatory bowel disease, acid gut syndrome,
liver failure and cirrhosis, failure of colostrum transfer in
neonates, ischemia (in any organ), bacteremia, infections within
body cavities such as the peritoneal, pericardial, thecal, and
pleural cavities, burns, severe wounds, excessive exercise or
stress, hemodialysis, conditions involving intolerable pain (e.g.,
pancreatitis, kidney stones), surgical operations, and non-healing
lesions. For these applications, the diagnostic methods of the
invention are typically used at a frequency that is effective to
monitor the early development of sepsis, inSIRS or post-surgical
inflammation to thereby enable early therapeutic intervention and
treatment of those conditions. In illustrative examples, the
diagnostic methods are used at least at 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hour
intervals or at least 1, 2, 3, 4, 5 or 6 day intervals, or at least
weekly, fortnightly or monthly.
[0041] Thus, in a related aspect, the present invention provides
methods for treating, preventing or inhibiting the development of
at least one condition selected from sepsis, inSIRS or
post-surgical inflammation in a subject. These methods generally
comprise: [0042] comparing the level of at least one IRC expression
product of a multi-transcript-producing gene in the subject to the
level of a corresponding IRC marker expression product in at least
one control subject selected from: a post-surgical
inflammation-positive subject, an inSIRS positive subject, and a
sepsis-positive subject, wherein a difference between the level of
the at least one IRC marker expression product and the level of the
corresponding IRC marker expression product indicates whether the
subject has, or is at risk of developing, one of the conditions,
wherein the at least one IRC marker expression product is
predetermined as being differentially expressed between at least
two of the conditions and wherein at least one other expression
product from the multi-transcript producing gene is predetermined
as being not so differentially expressed; and [0043] administering
to the subject, on the basis that the subject tests positive for
sepsis, an effective amount of an agent that treats or ameliorates
the symptoms or reverses or inhibits the development of sepsis, or
[0044] administering to the subject, on the basis that the subject
tests positive for inSIRS, an effective amount of an agent that
treats or ameliorates the symptoms or reverses or inhibits the
development of inSIRS; or [0045] administering to the subject, on
the basis that the subject tests positive for post-surgical
inflammation, an effective amount of an agent that treats or
ameliorates the symptoms or reverses or inhibits the development of
post-surgical inflammation.
[0046] Representative examples of sepsis treatments or agents
include but are not limited to, antibiotics, intravenous fluids,
vasoactives, palliative support for damaged or distressed organs
(e.g. oxygen for respiratory distress, fluids for hypovolemia) and
close monitoring of vital organs.
[0047] Non-limiting examples of such inSIRS treatments or agents
include but are not limited to, antibiotics, steroids, intravenous
fluids, glucocorticoids, vasoactives, palliative support for
damaged or distressed organs (e.g. oxygen for respiratory distress,
fluids for hypovolemia) and close monitoring of vital organs.
[0048] Illustrative examples of such post-surgical inflammation
treatments or agents include but are not limited to, antibiotics,
intravenous fluids, anti-inflammatory agents and immunomodulatory
agents.
[0049] Still another aspect of the present invention provides the
use of at least one IRC marker polynucleotide as broadly described
above, or at least one IRC marker polypeptide as broadly described
above, or at least one probe comprising or consisting essentially
of a nucleic acid sequence which corresponds or is complementary to
at least a portion of a nucleotide sequence encoding a IRC marker
polypeptide as broadly described above, or the use of at least one
antigen-binding molecule that is immuno-interactive with a IRC
marker polypeptide as broadly described above, in the manufacture
of a kit for assessing or diagnosing the presence or risk of
development of, or distinguishing between, sepsis, inSIRS and
post-surgical inflammation.
DETAILED DESCRIPTION OF THE INVENTION
1. Definitions
[0050] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0051] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0052] The term "differential expression," as used herein to
describe the expression of an IRC expression product of a
multi-transcript-producing gene, refers to the overexpression
(up-regulation) or underexpression (down-regulation) of the IRC
marker expression product (e.g., transcript or polypeptide)
relative to the level of expression of a corresponding IRC marker
expression product in a control subject as defined herein, and
encompasses a higher or lower level of a IRC marker expression
product (e.g., transcript or polypeptide) in a tissue sample or
body fluid relative to a reference sample. In certain embodiments,
an IRC marker expression product is differentially expressed if the
level of the IRC marker expression product in a biological sample
obtained from a test subject is at least 110%, 120%, 130%, 140%,
150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 600%, 700%,
800%, 900% or 1000%, or no more than about 95%, 90%, 80%, 70%, 60%,
50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%,
0.001% or 0.0001% of the level of expression of a corresponding IRC
marker gene expression product in a reference sample obtained from
a control subject as defined herein.
[0053] By "about" is meant a measurement, quantity, level, value,
number, frequency, percentage, dimension, size, amount, weight or
length that varies by as much as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%
to a reference measurement, quantity, level, value, number,
frequency, percentage, dimension, size, amount, weight or
length.
[0054] The term "amplicon" refers to a target sequence for
amplification, and/or the amplification products of a target
sequence for amplification. In certain other embodiments an
"amplicon" may include the sequence of probes or primers used in
amplification.
[0055] By "antigen-binding molecule" is meant a molecule that has
binding affinity for a target antigen. It will be understood that
this term extends to immunoglobulins, immunoglobulin fragments and
non-immunoglobulin derived protein frameworks that exhibit
antigen-binding activity.
[0056] As used herein, the term "binds specifically," "specifically
immuno-interactive" and the like when referring to an
antigen-binding molecule refers to a binding reaction which is
determinative of the presence of an antigen in the presence of a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified
antigen-binding molecules bind to a particular antigen and do not
bind in a significant amount to other proteins or antigens present
in the sample. Specific binding to an antigen under such conditions
may require an antigen-binding molecule that is selected for its
specificity for a particular antigen. For example, antigen-binding
molecules can be raised to a selected protein antigen, which bind
to that antigen but not to other proteins present in a sample. A
variety of immunoassay formats may be used to select
antigen-binding molecules specifically immuno-interactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immuno-interactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity.
[0057] The term "biological sample" as used herein refers to a
sample that may be extracted, untreated, treated, diluted or
concentrated from an animal. The biological sample may include a
biological fluid such as whole blood, serum, plasma, saliva, urine,
sweat, ascitic fluid, peritoneal fluid, synovial fluid, amniotic
fluid, cerebrospinal fluid, tissue biopsy, and the like. In certain
embodiments, the biological sample is blood, especially peripheral
blood.
[0058] Throughout this specification, unless the context requires
otherwise, the words "comprise," "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements:
[0059] By "corresponds to" or "corresponding to" is meant a
polynucleotide (a) having a nucleotide sequence that is
substantially identical or complementary to all or a portion of a
reference polynucleotide sequence or (b) encoding an amino acid
sequence identical to an amino acid sequence in a peptide or
protein. This phrase also includes within its scope a peptide or
polypeptide having an amino acid sequence that is substantially
identical to a sequence of amino acids in a reference peptide or
protein.
[0060] By "effective amount", in the context of treating or
preventing a condition is meant the administration of that amount
of active to an individual in need of such treatment or
prophylaxis, either in a single dose or as part of a series, that
is effective for the prevention of incurring a symptom, holding in
check such symptoms, and/or treating existing symptoms, of that
condition. The effective amount will vary depending upon the health
and physical condition of the individual to be treated, the
taxonomic group of individual to be treated, the formulation of the
composition, the assessment of the medical situation, and other
relevant factors. It is expected that the amount will fall in a
relatively broad range that can be determined through routine
trials.
[0061] The terms "expression" or "gene expression" refer to
production of RNA message or translation of RNA message into
proteins or polypeptides, or both. Detection of either types of
gene expression in use of any of the methods described herein is
encompassed by the present invention.
[0062] By "expression vector" is meant any autonomous genetic
element capable of directing the transcription of a polynucleotide
contained within the vector and suitably the synthesis of a peptide
or polypeptide encoded by the polynucleotide. Such expression
vectors are known to practitioners in the art.
[0063] As used herein, the term "functional activity" generally
refers to the ability of a molecule (e.g., a transcript or
polypeptide) to perform its designated function including a
biological, enzymatic, or therapeutic function. In certain
embodiments, the functional activity of a molecule corresponds to
its specific activity as determined by any suitable assay known in
the art.
[0064] The term "gene" as used herein refers to any and all
discrete coding regions of the cell's genome, as well as associated
non-coding and regulatory regions. The gene is also intended to
mean the open reading frame encoding specific polypeptides,
introns, and adjacent 5' and 3' non-coding nucleotide sequences
involved in the regulation of expression. In this regard, the gene
may further comprise control signals such as promoters, enhancers,
termination and/or polyadenylation signals that are naturally
associated with a given gene, or heterologous control signals. The
DNA sequences may be cDNA or genomic DNA or a fragment thereof. The
gene may be introduced into an appropriate vector for
extrachromosomal maintenance or for integration into the host.
[0065] By "high density polynucleotide arrays" and the like is
meant those arrays that contain at least 400 different features per
cm.sup.2.
[0066] The phrase "high discrimination hybridization conditions"
refers to hybridization conditions in which single base mismatch
may be determined.
[0067] By "housekeeping gene" is meant a gene that is expressed in
virtually all cells since it is fundamental to the any cell's
functions (e.g., essential proteins and RNA molecules).
[0068] "Hybridization" is used herein to denote the pairing of
complementary nucleotide sequences to produce a DNA-DNA hybrid or a
DNA-RNA hybrid. Complementary base sequences are those sequences
that are related by the base-pairing rules. In DNA, A pairs with T
and C pairs with G. In RNA, U pairs with A and C pairs with G. In
this regard, the terms "match" and "mismatch" as used herein refer
to the hybridization potential of paired nucleotides in
complementary nucleic acid strands. Matched nucleotides hybridize
efficiently, such as the classical A-T and G-C base pair mentioned
above. Mismatches are other combinations of nucleotides that do not
hybridize efficiently.
[0069] The phrase "hybridizing specifically to" and the like refer
to the binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA.
[0070] Reference herein to "immuno-interactive" includes reference
to any interaction, reaction, or other form of association between
molecules and in particular where one of the molecules is, or
mimics, a component of the immune system.
[0071] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated polynucleotide," as used
herein, refers to a polynucleotide, which has been purified from
the sequences which flank it in a naturally-occurring state, e.g.,
a DNA fragment which has been removed from the sequences that are
normally adjacent to the fragment. Alternatively, an "isolated
peptide" or an "isolated polypeptide" and the like, as used herein,
refer to in vitro isolation and/or purification of a peptide or
polypeptide molecule from its natural cellular environment, and
from association with other components of the cell, i.e., it is not
associated with in vivo substances.
[0072] As used herein, a "naturally-occurring" nucleic acid
molecule refers to a RNA or DNA molecule having a nucleotide
sequence that occurs in nature. For example a naturally-occurring
nucleic acid molecule can encode a protein that occurs in
nature.
[0073] By "obtained" is meant to come into possession. Biological
or reference samples so obtained include, for example, nucleic acid
extracts or polypeptide extracts isolated or derived from a
particular source. For instance, the extract may be isolated
directly from a biological fluid or tissue of a subject.
[0074] The term "oligonucleotide" as used herein refers to a
polymer composed of a multiplicity of nucleotide residues
(deoxyribonucleotides or ribonucleotides, or related structural
variants or synthetic analogues thereof, including nucleotides with
modified or substituted sugar groups and the like) linked via
phosphodiester bonds (or related structural variants or synthetic
analogues thereof). Thus, while the term "oligonucleotide"
typically refers to a nucleotide polymer in which the nucleotide
residues and linkages between them are naturally-occurring, it will
be understood that the term also includes within its scope various
analogues including, but not restricted to, peptide nucleic acids
(PNAs), phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phosphoraniladate,
phosphoroamidate, methyl phosphonates, 2-O-methyl ribonucleic
acids, and the like. The exact size of the molecule can vary
depending on the particular application. Oligonucleotides are a
polynucleotide subset with 200 bases or fewer in length.
Preferably, oligonucleotides are 10 to 60 bases in length and most
preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in
length. Oligonucleotides are usually single stranded, e.g., for
probes; although oligonucleotides may be double stranded, e.g., for
use in the construction of a variant nucleic acid sequence.
Oligonucleotides of the invention can be either sense or antisense
oligonucleotides.
[0075] The term "oligonucleotide array" refers to a substrate
having oligonucleotide probes with different known sequences
deposited at discrete known locations associated with its surface.
For example, the substrate can be in the form of a two dimensional
substrate as described in U.S. Pat. No. 5,424,186. Such substrate
may be used to synthesize two-dimensional spatially addressed
oligonucleotide (matrix) arrays. Alternatively, the substrate may
be characterized in that it forms a tubular array in which a two
dimensional planar sheet is rolled into a three-dimensional tubular
configuration. The substrate may also be in the form of a
microsphere or bead connected to the surface of an optic fiber as,
for example, disclosed by Chee et al. in WO 00/39587.
Oligonucleotide arrays have at least two different features and a
density of at least 400 features per cm.sup.2. In certain
embodiments, the arrays can have a density of about 500, at least
one thousand, at least 10 thousand, at least 100 thousand, at least
one million or at least 10 million features per cm.sup.2. For
example, the substrate may be silicon or glass and can have the
thickness of a glass microscope slide or a glass cover slip, or may
be composed of other synthetic polymers. Substrates that are
transparent to light are useful when the method of performing an
assay on the substrate involves optical detection. The term also
refers to a probe array and the substrate to which it is attached
that form part of a wafer.
[0076] The term "operably connected" or "operably linked" as used
herein means placing a structural gene under the regulatory control
of a promoter, which then controls the transcription and optionally
translation of the gene. In the construction of heterologous
promoter/structural gene combinations, it is generally preferred to
position the genetic sequence or promoter at a distance from the
gene transcription start site that is approximately the same as the
distance between that genetic sequence or promoter and the gene it
controls in its natural setting; i.e. the gene from which the
genetic sequence or promoter is derived. As is known in the art,
some variation in this distance can be accommodated without loss of
function. Similarly, the preferred positioning of a regulatory
sequence element with respect to a heterologous gene to be placed
under its control is defined by the positioning of the element in
its natural setting; i.e., the genes from which it is derived.
[0077] The term "pathogen" is used herein in its broadest sense to
refer to an organism or an infectious agent whose infection of
cells of viable animal tissue elicits a disease response.
[0078] The term "polynucleotide" or "nucleic acid" as used herein
designates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers
to a polymeric form of nucleotides of at least 10 bases in length,
either ribonucleotides or deoxynucleotides or a modified form of
either type of nucleotide. The term includes single and double
stranded forms of DNA.
[0079] The terms "polynucleotide variant" and "variant" refer to
polynucleotides displaying substantial sequence identity with a
reference polynucleotide sequence or polynucleotides that hybridize
with a reference sequence under stringent conditions that are
defined hereinafter. These terms also encompass polynucleotides in
which one or more nucleotides have been added or deleted, or
replaced with different nucleotides. In this regard, it is well
understood in the art that certain alterations inclusive of
mutations, additions, deletions and substitutions can be made to a
reference polynucleotide whereby the altered polynucleotide retains
a biological function or activity of the reference polynucleotide.
The terms "polynucleotide variant" and "variant" also include
naturally-occurring allelic variants.
[0080] "Polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid residues
and to variants and synthetic analogues of the same. Thus, these
terms apply to amino acid polymers in which one or more amino acid
residues is a synthetic non-naturally-occurring amino acid, such as
a chemical analogue of a corresponding naturally-occurring amino
acid, as well as to naturally-occurring amino acid polymers.
[0081] The term "polypeptide variant" refers to polypeptides which
are distinguished from a reference polypeptide by the addition,
deletion or substitution of at least one amino acid residue. In
certain embodiments, one or more amino acid residues of a reference
polypeptide are replaced by different amino acids. It is well
understood in the art that some amino acids may be changed to
others with broadly similar properties without changing the nature
of the activity of the polypeptide (conservative substitutions) as
described hereinafter.
[0082] As used herein, "post-surgical inflammation" refers to a
condition arising due to an immune response to a stimulus relating
to a surgical insult. Post-surgical inflammation can be local or
systemic and is often characterized by swelling, fever, pain and/or
redness. Inflammation involves the movement of fluid and cells
(e.g., white blood cells or leukocytes, neutrophils, monocytes and
T- and B-cells) into the affected area, site or tissue. Excessive,
misdirected and/or inappropriate immune inflammatory responses
resulting from surgery can lead to SIRS and to damage of normal,
healthy body tissues.
[0083] By "primer" is meant an oligonucleotide which, when paired
with a strand of DNA, is capable of initiating the synthesis of a
primer extension product in the presence of a suitable polymerizing
agent. The primer is preferably single-stranded for maximum
efficiency in amplification but can alternatively be
double-stranded. A primer must be sufficiently long to prime the
synthesis of extension products in the presence of the
polymerization agent. The length of the primer depends on many
factors, including application, temperature to be employed,
template reaction conditions, other reagents, and source of
primers. For example, depending on the complexity of the target
sequence, the primer may be at least about 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, to one base
shorter in length than the template sequence at the 3' end of the
primer to allow extension of a nucleic acid chain, though the 5'
end of the primer may extend in length beyond the 3' end of the
template sequence. In certain embodiments, primers can be large
polynucleotides, such as from about 35 nucleotides to several
kilobases or more. Primers can be selected to be "substantially
complementary" to the sequence on the template to which it is
designed to hybridize and serve as a site for the initiation of
synthesis. By "substantially complementary", it is meant that the
primer is sufficiently complementary to hybridize with a target
polynucleotide. Desirably, the primer contains no mismatches with
the template to which it is designed to hybridize but this is not
essential. For example, non-complementary nucleotide residues can
be attached to the 5' end of the primer, with the remainder of the
primer sequence being complementary to the template. Alternatively,
non-complementary nucleotide residues or a stretch of
non-complementary nucleotide residues can be interspersed into a
primer, provided that the primer sequence has sufficient
complementarity with the sequence of the template to hybridize
therewith and thereby form a template for synthesis of the
extension product of the primer.
[0084] "Probe" refers to a molecule that binds to a specific
sequence or sub-sequence or other moiety of another molecule.
Unless otherwise indicated, the term "probe" typically refers to a
polynucleotide probe that binds to another polynucleotide, often
called the "target polynucleotide", through complementary base
pairing. Probes can bind target polynucleotides lacking complete
sequence complementarity with the probe, depending on the
stringency of the hybridization conditions. Probes can be labeled
directly or indirectly and include primers within their scope.
[0085] The term "recombinant polynucleotide" as used herein refers
to a polynucleotide formed in vitro by the manipulation of nucleic
acid into a form not normally found in nature. For example, the
recombinant polynucleotide may be in the form of an expression
vector. Generally, such expression vectors include transcriptional
and translational regulatory nucleic acid operably linked to the
nucleotide sequence.
[0086] By "recombinant polypeptide" is meant a polypeptide made
using recombinant techniques, i.e., through the expression of a
recombinant or synthetic polynucleotide.
[0087] By "regulatory element" or "regulatory sequence" is meant
nucleic acid sequences (e.g., DNA) necessary for expression of an
operably linked coding sequence in a particular host cell. The
regulatory sequences that are suitable for prokaryotic cells for
example, include a promoter, and optionally a cis-acting sequence
such as an operator sequence and a ribosome binding site. Control
sequences that are suitable for eukaryotic cells include promoters,
polyadenylation signals, transcriptional enhancers, translational
enhancers, leader or trailing sequences that modulate mRNA
stability, as well as targeting sequences that target a product
encoded by a transcribed polynucleotide to an intracellular
compartment within a cell or to the extracellular environment.
[0088] As used herein, "sepsis" is defined as SIRS with a presumed
or confirmed systemic infectious process. Confirmation of
infectious process can be determined using microbiological culture
or isolation of the infectious agent. From an immunological
perspective, sepsis may be seen as a systemic response to systemic
live microorganisms or systemic infection.
[0089] The term "sequence identity" as used herein refers to the
extent that sequences are identical on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity" is calculated
by comparing two optimally aligned sequences over the window of
comparison, determining the number of positions at which the
identical nucleic acid base (e.g., A, T, C, G, I) or the identical
amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile,
Phe, Tyr, Trp, Lys, Arg, H is, Asp, Glu, Asn, Gln, Cys and Met)
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. For the purposes of the present invention, "sequence
identity" will be understood to mean the "match percentage"
calculated by the DNASIS computer program (Version 2.5 for windows;
available from Hitachi Software engineering Co., Ltd., South San
Francisco, Calif., USA) using standard defaults as used in the
reference manual accompanying the software.
[0090] "Similarity" refers to the percentage number of amino acids
that are identical or constitute conservative substitutions as
defined in Table A infra. Similarity may be determined using
sequence comparison programs such as GAP (Deveraux et al. 1984,
Nucleic Acids Research 12, 387-395). In this way, sequences of a
similar or substantially different length to those cited herein
might be compared by insertion of gaps into the alignment, such
gaps being determined, for example, by the comparison algorithm
used by GAP.
[0091] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence,"
"comparison window," "sequence identity," "percentage of sequence
identity" and "substantial identity". A "reference sequence" is at
least 12 but frequently 15 to 18 and often at least 25 monomer
units, inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.,
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
at least 6 contiguous positions, usually about 50 to about 100,
more usually about 100 to about 150 in which a sequence is compared
to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned. The comparison
window may comprise additions or deletions (i.e., gaps) of about
20% or less as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences. Optimal alignment of sequences for aligning a comparison
window may be conducted by computerized implementations of
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package Release 7.0, Genetics Computer Group, 575
Science Drive Madison, Wis., USA) or by inspection and the best
alignment (i.e., resulting in the highest percentage homology over
the comparison window) generated by any of the various methods
selected. Reference also may be made to the BLAST family of
programs as for example disclosed by Altschul et al., 1997, Nucl.
Acids Res. 25:3389. A detailed discussion of sequence analysis can
be found in Unit 19.3 of Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley & Sons Inc, 1994-1998, Chapter
15.
[0092] The terms "subject" or "individual" or "patient", used
interchangeably herein, refer to any subject, particularly a
vertebrate subject, and even more particularly a mammalian subject,
for whom therapy or prophylaxis is desired. Suitable vertebrate
animals that fall within the scope of the invention include, but
are not restricted to, primates, avians, livestock animals (e.g.,
sheep, cows, horses, donkeys, pigs), laboratory test animals (e.g.,
rabbits, mice, rats, guinea pigs, hamsters), companion animals
(e.g., cats, dogs) and captive wild animals (e.g., foxes, deer,
dingoes). A preferred subject is an equine animal in need of
treatment or prophylaxis of sepsis. However, it will be understood
that the aforementioned terms do not imply that symptoms are
present.
[0093] The phrase "substantially similar affinities" refers herein
to target sequences having similar strengths of detectable
hybridization to their complementary or substantially complementary
oligonucleotide probes under a chosen set of stringent
conditions.
[0094] "Systemic Inflammatory Response Syndrome (SIRS)," as used
herein, refers to a clinical response arising from a non-specific
insult with two or more of the following measurable clinical
characteristics; a body temperature greater than 38.degree. C. or
less than 36.degree. C., a heart rate greater than 90 beats per
minute, a respiratory rate greater than 20 per minute, a white
blood cell count (total leukocytes) greater than 12,000 per
mm.sup.3 or less than 4,000 per mm.sup.3, or a band neutrophil
percentage greater than 10%. From an immunological perspective, it
may be seen as representing a systemic response to insult (e.g.,
major surgery) or systemic inflammation. As used herein, therefore,
"infection-negative SIRS (inSIRS)" includes the clinical response
noted above but in the absence of a systemic infectious
process.
[0095] The term "template" as used herein refers to a nucleic acid
that is used in the creation of a complementary nucleic acid strand
to the "template" strand. The template may be either RNA and/or
DNA, and the complementary strand may also be RNA and/or DNA. In
certain embodiments, the complementary strand may comprise all or
part of the complementary sequence to the "template," and/or may
include mutations so that it is not an exact, complementary strand
to the "template". Strands that are not exactly complementary to
the template strand may hybridize specifically to the template
strand in detection assays described here, as well as other assays
known in the art, and such complementary strands that can be used
in detection assays are part of the invention.
[0096] The term "transformation" means alteration of the genotype
of an organism, for example a bacterium, yeast, mammal, avian,
reptile, fish or plant, by the introduction of a foreign or
endogenous nucleic acid.
[0097] The term "treat" is meant to include both therapeutic and
prophylactic treatment.
[0098] By "vector" is meant a polynucleotide molecule, suitably a
DNA molecule derived, for example, from a plasmid, bacteriophage,
yeast, virus, mammal, avian, reptile or fish into which a
polynucleotide can be inserted or cloned. A vector preferably
contains one or more unique restriction sites and can be capable of
autonomous replication in a defined host cell including a target
cell or tissue or a progenitor cell or tissue thereof, or be
integrable with the genome of the defined host such that the cloned
sequence is reproducible. Accordingly, the vector can be an
autonomously replicating vector, i.e., a vector that exists as an
extrachromosomal entity, the replication of which is independent of
chromosomal replication, e.g., a linear or closed circular plasmid,
an extrachromosomal element, a minichromosome, or an artificial
chromosome. The vector can contain any means for assuring
self-replication. Alternatively, the vector can be one which, when
introduced into the host cell, is integrated into the genome and
replicated together with the chromosome(s) into which it has been
integrated. A vector system can comprise a single vector or
plasmid, two or more vectors or plasmids, which together contain
the total DNA to be introduced into the genome of the host cell, or
a transposon. The choice of the vector will typically depend on the
compatibility of the vector with the host cell into which the
vector is to be introduced. The vector can also include a selection
marker such as an antibiotic resistance gene that can be used for
selection of suitable transformants. Examples of such resistance
genes are known to those of skill in the art.
[0099] The terms "wild-type" and "normal" are used interchangeably
to refer to the phenotype that is characteristic of most of the
members of the species occurring naturally and contrast for example
with the phenotype of a mutant.
2. Abbreviations
[0100] The following abbreviations are used throughout the
application:
[0101] nt=nucleotide
[0102] nts=nucleotides
[0103] aa=amino acid(s)
[0104] kb=kilobase(s) or kilobase pair(s)
[0105] kDa=kilodalton(s)
[0106] d=day
[0107] h=hour
[0108] s=seconds
3. Markers of Sepsis, inSIRS and Post-Surgical Inflammation and
Uses Therefor
[0109] The present invention is predicated in part on the
identification of 235 genes that show evidence of splice variation
in which only particular splice variants of individual genes differ
in expression between sepsis-positive patients, inSIRS-positive
patients and post-surgical patients. Of these 235
multi-transcript-producing genes, only a limited number (57) were
found to express specific splice variants, which comprise
"condition-separating exons" and which are useful as classifiers to
distinguish between these patient groups. These
multi-transcript-producing genes are listed in Table 1.
[0110] Thus, in accordance with the present invention, specific
splice variants of the above multi-transcript-producing genes and
their polypeptide products provide a means for separating sepsis,
inSIRS and post-surgical inflammation, allowing for qualitative or
quantitative grading of inflammatory response as if there were a
"continuum" of severity of inflammatory response from post-surgical
inflammation through to sepsis. These markers are thus designated
herein "inflammatory response continuum" or "IRC" marker expression
products, which are listed in Table 2, 3 and 4.
[0111] Accordingly, the IRC marker expression products of the
present invention are useful in methods for diagnosis, detection of
host response, determining degree of host response, monitoring,
treatment or management of, or distinguishing between,
infection-negative systemic inflammatory response syndrome (inSIRS)
and sepsis as well as post-surgical inflammation in mammals. More
particularly, the present invention relates to the use of specific
expression products from a multi-transcript-producing gene for
distinguishing between inSIRS and sepsis and post-surgical
inflammation.
[0112] In specific embodiments, the IRC markers are in the form of
RNA molecules of specified sequences, or polypeptides transcribed
from these RNA molecules in cells, especially in blood cells, and
more especially in peripheral blood cells, of subjects with or
susceptible to sepsis/inSIRS/post-surgical inflammation, are
disclosed. These markers are indicators of
sepsis/inSIRS/post-surgical inflammation and, when differentially
expressed as compared to their expression in control subjects
selected from sepsis-positive subjects, inSIRS-positive subjects,
post-surgical inflammation positive subjects and normal subjects or
subjects that do not have any of these conditions, they distinguish
between, and are diagnostic for the presence or absence of, those
conditions in tested subjects. Such markers provide considerable
advantages over the prior art in this field. In certain
advantageous embodiments where leukocytes (e.g., peripheral blood
cells) are used for the analysis, it is possible to diagnose sepsis
before serum antibodies to endotoxin, or endotoxemia-causing agents
are detected.
[0113] It will be apparent that the nucleic acid sequences
disclosed herein (also referred to herein as "IRC marker
polynucleotides") will find utility in a variety of applications in
detection, diagnosis, prognosis and treatment of sepsis, inSIRS and
post-surgical inflammation. Examples of such applications within
the scope of the present disclosure include amplification of IRC
marker polynucleotides using specific primers, detection of IRC
marker polynucleotides by hybridization with oligonucleotide
probes, incorporation of isolated nucleic acids into vectors,
expression of vector-incorporated nucleic acids as RNA and protein,
and development of immunological/detection/diagnostic/prognostic
reagents corresponding to marker encoded products.
[0114] The identified IRC marker polynucleotides may in turn be
used to design specific oligonucleotide probes and primers. Such
probes and primers may be of any length that would specifically
hybridize to the identified IRC marker polynucleotides and may be
at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300,
400, 500 nucleotides in length and in the case of probes, up to the
full-length of the sequences of one or more of condition-separating
exons contained in a IRC marker polynucleotide or up to the
full-length of an IRC marker polynucleotide as identified herein.
Probes may, also include additional sequence at their 5' and/or 3'
ends so that they extent beyond the target sequence with which they
hybridize.
[0115] When used in combination with nucleic acid amplification
procedures, these probes and primers enable the rapid analysis of
biological samples (e.g., peripheral blood samples) for detecting
or quantifying IRC marker polynucleotides (e.g., transcripts). Such
procedures include any method or technique known in the art or
described herein for duplicating or increasing the number of copies
or amount of a target nucleic acid or its complement.
[0116] One of ordinary skill in the art could select segments from
the identified IRC marker polynucleotides and their encoded
polypeptide products (also referred to herein as "IRC marker
polypeptides") for use in the different detection, diagnostic, or
prognostic methods, vector constructs, antigen-binding molecule
production, kit, and/or any of the embodiments described herein as
part of the present invention. Representative sequences that are
desirable for use in the invention are those set forth in SEQ ID
NO: 1-88 (see Tables 2, 3 and 4).
4. Nucleic Acid Molecules of the Invention
[0117] As described in the Examples and in Tables 1-4, the present
disclosure provides IRC marker polynucleotides comprising
condition-separating exons from 57 multi-transcript-producing genes
selected from ANKDD1A, GABRR2, OTX1, PANX2, RHBDF2, SLAMF7, AMBRA1,
CES2, CLPB, HIPK2, C1ORF91, NDST1, SLC36A1, ADAM19, CUL7, TG,
PDCD1LG2, GRINL1A, MGRN1, SNTB2, CDK5R1, GAA, KATNAL2, CEACAM4,
ZNF335, ASPHD2, ACRC, BTNL8, MOV10, MED12L, KLHL6, PDLIM5, GALNT10,
SCRN1, VOPP1, FKBP9, KIF27, PIWIL4, TEP1, GCH1, PRR11, CDH2, PPM1N,
RRAS, DDOST, APH1A, TTL, TEX261, COQ2, FCHSD1, BAK1, SLC25A25,
RELT, ACP2, TBC1D2B, FANCA or SLC39A11. Representative IRC marker
polynucleotides have been identified by exon array analysis of
blood obtained from patients with clinical evidence of sepsis or
inSIRS or post-surgical inflammation and these are set forth in SEQ
ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,
67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,
101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,
127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,
153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,
179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203,
205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229,
231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255,
257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281,
283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307,
309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333,
335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359,
361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385,
387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411,
413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437,
439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463,
465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489,
491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513 or 515.
These sequences, which are presented in Tables 2-4, are diagnostic
for the presence or absence of sepsis or inSIRS or post-surgical
inflammation.
[0118] In accordance with the present invention, the sequences of
isolated nucleic acids disclosed herein find utility inter alia as
hybridization probes or amplification primers. In certain
embodiments, these probes and primers represent oligonucleotides,
which are of sufficient length to provide specific hybridization to
a RNA or DNA sample extracted from the biological sample. The
sequences typically will be about 10-20 nucleotides, but may be
longer. Longer sequences, e.g., of about 30, 40, 50, 100, 500 and
even up to the full-length of condition-separating exons or of the
IRC marker polynucleotides, are desirable for certain
embodiments.
[0119] Nucleic acid molecules having contiguous stretches of about
10, 15, 17, 20, 30, 40, 50, 60, 75 or 100 or 500 nucleotides of a
sequence set forth in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,
191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,
217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,
243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,
269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,
295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,
321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,
347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371,
373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397,
399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,
425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449,
451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475,
477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,
503, 505, 507, 509, 511, 513 or 515 are contemplated. Molecules
that are complementary to the above mentioned sequences and that
bind to these sequences under high stringency conditions are also
contemplated. These probes are useful in a variety of hybridization
embodiments, such as Southern and northern blotting. In some cases,
it is contemplated that probes may be used that hybridize to
multiple target sequences without compromising their ability to
effectively diagnose the presence or absence or distinguish between
sepsis, inSIRS and post-surgical inflammation. In general, it is
contemplated that the hybridization probes described herein are
useful both as reagents in solution hybridization, as in PCR, for
detection of expression of corresponding genes, as well as in
embodiments employing a solid phase.
[0120] Various probes and primers may be designed around the
disclosed nucleotide sequences. For example, in certain
embodiments, the sequences used to design probes and primers may
include repetitive stretches of adenine nucleotides (poly-A tails)
normally attached at the ends of the RNA for the identified marker
genes. In other embodiments, probes and primers may be specifically
designed to not include these or other segments from the identified
marker genes, as one of ordinary skilled in the art may deem
certain segments more suitable for use in the detection methods
disclosed. In any event, the choice of primer or probe sequences
for a selected application is within the realm of the ordinary
skilled practitioner. Illustrative primer/probe sequences for
detection of IRC marker polynucleotides are presented in Table
5.
[0121] Primers may be provided in double-stranded or
single-stranded form, although the single-stranded form is
desirable. Probes, while perhaps capable of priming, are designed
to bind to a target DNA or RNA and need not be used in an
amplification process. In certain embodiments, the probes or
primers are labeled with radioactive species .sup.32P, .sup.14C,
.sup.35S, .sup.3H, or other label), with a fluorophore (e.g.,
rhodamine, fluorescein) or with a chemillumiscent label (e.g.,
luciferase).
[0122] The present invention provides substantially full-length
cDNA sequences that are useful as markers of sepsis, inSIRS and
post-surgical inflammation. It will be understood, however, that
the present disclosure is not limited to these disclosed sequences
and is intended particularly to encompass at least isolated nucleic
acids that are hybridizable to nucleic acids comprising the
disclosed sequences or that are variants of these nucleic acids.
For example, a nucleic acid of partial sequence may be used to
identify a structurally-related gene or the full-length genomic or
cDNA clone from which it is derived. Methods for generating cDNA
and genomic libraries which may be used as a target for the
above-described probes are known in the art (see, for example,
Sambrook et al., 1989, supra and Ausubel et al., 1994, supra). All
such nucleic acids as well as the specific nucleic acid molecules
disclosed herein are collectively referred to as "IRC marker
polynucleotides." Additionally, the present invention includes
within its scope isolated or purified polypeptide products of IRC
marker polynucleotides.
[0123] As such, the present invention encompasses isolated or
substantially purified nucleic acid or protein compositions. An
"isolated" or "purified" nucleic acid molecule or protein, or
biologically active portion thereof, is substantially or
essentially free from components that normally accompany or
interact with the nucleic acid molecule or protein as found in its
naturally occurring environment. Thus, an isolated or purified
polynucleotide or polypeptide is substantially free of other
cellular material, or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. Suitably, an "isolated"
polynucleotide is free of sequences (especially protein encoding
sequences) that naturally flank the polynucleotide (i.e., sequences
located at the 5' and 3' ends of the polynucleotide) in the genomic
DNA of the organism from which the polynucleotide was derived. For
example, in various embodiments, an isolated IRC marker
polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb,
1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally
flank the polynucleotide in genomic DNA of the cell from which the
polynucleotide was derived. A polypeptide that is substantially
free of cellular material includes preparations of protein having
less than about 30%, 20%, 10%, 5%, (by dry weight) of contaminating
protein. When the IRC marker polypeptide is recombinantly produced,
culture medium suitably represents less than about 30%, 20%, 10%,
or 5% (by dry weight) of chemical precursors or
non-protein-of-interest chemicals.
[0124] The invention also contemplates variants of the IRC marker
polynucleotides. Nucleic acid variants can be naturally-occurring,
such as allelic variants (same locus), homologues (different
locus), and orthologues (different organism) or can be non
naturally-occurring. Naturally occurring variants such as these can
be identified with the use of well-known molecular biology
techniques, as, for example, with polymerase chain reaction (PCR)
and hybridization techniques as known in the art. Non-naturally
occurring variants can be made by mutagenesis techniques, including
those applied to polynucleotides, cells, or organisms. The variants
can contain nucleotide substitutions, deletions, inversions and
insertions. Variation can occur in either or both the coding and
non-coding regions. The variations can produce both conservative
and non-conservative amino acid substitutions (as compared in the
encoded product). For nucleotide sequences, conservative variants
include those sequences that, because of the degeneracy of the
genetic code, encode the amino acid sequence of one of the IRC
marker polypeptides of the invention. Variant nucleotide sequences
also include synthetically derived nucleotide sequences, such as
those generated, for example, by using site-directed mutagenesis
but which still encode an IRC marker polypeptide of the invention.
Generally, variants of a particular nucleotide sequence of the
invention will have at least about 70%, 75%, 80%, 85%, desirably
about 90%, 91%, 92%, 93%, 94% to 95% or more, and more suitably
about 96%, 97%, 98%, 99% or more sequence identity to that
particular nucleotide sequence as determined by sequence alignment
programs described elsewhere herein using default parameters.
[0125] The IRC marker polynucleotides of the invention can be used
to isolate corresponding sequences and alleles from other
organisms, particularly other mammals. Methods are readily
available in the art for the hybridization of nucleic acid
sequences. Coding sequences from other organisms may be isolated
according to well known techniques based on their sequence identity
with the coding sequences set forth herein. In these techniques all
or part of the known coding sequence is used as a probe which
selectively hybridizes to other IRC marker polynucleotide coding
sequences present in a population of cloned cDNA fragments (i.e.,
cDNA libraries) from a chosen organism. Accordingly, the present
invention also contemplates polynucleotides that hybridize to the
IRC marker polynucleotide sequences, or to their complements, under
stringency conditions described below. As used herein, the term
"hybridizes under low stringency, medium stringency, high
stringency, or very high stringency conditions" describes
conditions for hybridization and washing. Guidance for performing
hybridization reactions can be found in Ausubel et al., (1998,
supra), Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are
described in that reference and either can be used. Reference
herein to low stringency conditions include and encompass from at
least about 1% v/v to at least about 15% v/v formamide and from at
least about 1 M to at least about 2 M salt for hybridization at
42.degree. C., and at least about 1 M to at least about 2 M salt
for washing at 42.degree. C. Low stringency conditions also may
include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO.sub.4
(pH 7.2), 7% SDS for hybridization at 65.degree. C., and (i)
2.times.SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM
NaHPO.sub.4 (pH 7.2), 5% SDS for washing at room temperature. One
embodiment of low stringency conditions includes hybridization in
6.times. sodium chloride/sodium citrate (SSC) at about 45.degree.
C., followed by two washes in 0.2.times.SSC, 0.1% SDS at least at
50.degree. C. (the temperature of the washes can be increased to
55.degree. C. for low stringency conditions). Medium stringency
conditions include and encompass from at least about 16% v/v to at
least about 30% v/v formamide and from at least about 0.5 M to at
least about 0.9 M salt for hybridization at 42.degree. C., and at
least about 0.1 M to at least about 0.2 M salt for washing at
55.degree. C. Medium stringency conditions also may include 1%
Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2),
7% SDS for hybridization at 65.degree. C., and (i) 2.times.SSC,
0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2),
5% SDS for washing at 60-65.degree. C. One embodiment of medium
stringency conditions includes hybridizing in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C. High stringency conditions include and
encompass from at least about 31% v/v to at least about 50% v/v
formamide and from about 0.01 M to about 0.15 M salt for
hybridization at 42.degree. C., and about 0.01 M to about 0.02 M
salt for washing at 55.degree. C. High stringency conditions also
may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO.sub.4 (pH 7.2), 7% SDS
for hybridization at 65.degree. C., and (i) 0.2.times.SSC, 0.1%
SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO.sub.4 (pH 7.2), 1%
SDS for washing at a temperature in excess of 65.degree. C. One
embodiment of high stringency conditions includes hybridizing in
6.times.SSC at about 45.degree. C., followed by one or more washes
in 0.2.times.SSC, 0.1% SDS at 65.degree. C.
[0126] In certain embodiments, an IRC marker polynucleotide of the
invention is encoded by a polynucleotide that hybridizes to a
disclosed nucleotide sequence (and suitably comprises a
condition-separating exon as defined herein) under very high
stringency conditions. One embodiment of very high stringency
conditions includes hybridizing 0.5 M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C.
[0127] Other stringency conditions are well known in the art and a
skilled addressee will recognize that various factors can be
manipulated to optimize the specificity of the hybridization.
Optimization of the stringency of the final washes can serve to
ensure a high degree of hybridization. For detailed examples, see
Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et
al. (1989, supra) at sections 1.101 to 1.104.
[0128] While stringent washes are typically carried out at
temperatures from about 42.degree. C. to 68.degree. C., one skilled
in the art will appreciate that other temperatures may be suitable
for stringent conditions. Maximum hybridization rate typically
occurs at about 20.degree. C. to 25.degree. C. below the T.sub.m
for formation of a DNA-DNA hybrid. It is well known in the art that
the T.sub.m is the melting temperature, or temperature at which two
complementary polynucleotide sequences dissociate. Methods for
estimating T.sub.m are well known in the art (see Ausubel et al.,
supra at page 2.10.8). In general, the T.sub.m of a perfectly
matched duplex of DNA may be predicted as an approximation by the
formula:
T.sub.m=81.5+16.6(log.sub.10 M)+0.41(% G+C)-0.63(%
formamide)-(600/length)
[0129] wherein: M is the concentration of Na.sup.+, preferably in
the range of 0.01 molar to 0.4 molar; % G+C is the sum of guanosine
and cytosine bases as a percentage of the total number of bases,
within the range between 30% and 75% G+C; % formamide is the
percent formamide concentration by volume; length is the number of
base pairs in the DNA duplex. The T.sub.m of a duplex DNA decreases
by approximately 1.degree. C. with every increase of 1% in the
number of randomly mismatched base pairs. Washing is generally
carried out at T.sub.m-15.degree. C. for high stringency, or
T.sub.m-30.degree. C. for moderate stringency.
[0130] In one example of a hybridization procedure, a membrane
(e.g., a nitrocellulose membrane or a nylon membrane) containing
immobilized DNA is hybridized overnight at 42.degree. C. in a
hybridization buffer (50% deionized formamide, 5.times.SSC,
5.times.Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrrolidone
and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured
salmon sperm DNA) containing labeled probe. The membrane is then
subjected to two sequential medium stringency washes (i.e.,
2.times.SSC, 0.1% SDS for 15 min at 45.degree. C., followed by
2.times.SSC, 0.1% SDS for 15 min at 50.degree. C.), followed by two
sequential higher stringency washes (i.e., 0.2.times.SSC, 0.1% SDS
for 12 min at 55.degree. C. followed by 0.2.times.SSC and 0.1% SDS
solution for 12 min at 65-68.degree. C.
5. Polypeptides of the Invention
[0131] The present invention also contemplates the use of
full-length polypeptides encoded by the IRC marker polynucleotides
of the invention as well as their fragments, which are referred to
collectively herein as "IRC marker polypeptides" for use as
positive controls in the methods of the invention. Fragments of
full-length IRC marker polypeptides include amino acid sequences
encoded by condition-separating exons as defined herein and may
comprise 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 60 amino
acid residues in length. For example, fragments contemplated by the
present invention are at least 6 and desirably at least 8 amino
acid residues in length, which can elicit an immune response in an
animal for the production of antigen-binding molecules that are
immuno-interactive with an IRC marker polypeptide of the invention.
Such antigen-binding molecules can be used to screen vertebrate
animals, especially mammals, for structurally and/or functionally
related IRC marker polypeptides: Fragments of a full-length IRC
marker polypeptide include peptides comprising amino acid sequences
sufficiently similar to or derived from the amino acid sequences of
a (putative) full-length IRC marker polypeptide, for example, the
amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14,
16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,
50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,
84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,
114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,
140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,
166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190,
192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,
218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,
244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,
270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294,
296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320,
322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346,
348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372,
374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398,
400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424,
426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450,
452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476,
478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502,
504, 506, 508, 510, 512, 514 or 516, which include less amino acids
than a full-length IRC marker polypeptide. A fragment of a
full-length IRC marker polypeptide can be a polypeptide which is,
for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150,
300, 400, 500, 600, 700, 800, 900 or 1000, or even at least about
2000 or 3000, or more amino acid residues in length.
[0132] The present invention also contemplates detecting variant
IRC marker polypeptides, which comprise an amino acid sequence
encoded by a condition-separating exon or variant thereof, in the
methods of the invention. "Variant" polypeptides include proteins
derived from the native protein by deletion (so-called truncation)
or addition of one or more amino acids to the N-terminal and/or
C-terminal end of the native protein; deletion or addition of one
or more amino acids at one or more sites in the native protein; or
substitution of one or more amino acids at one or more sites in the
native protein. Variant proteins encompassed by the present
invention are biologically active, that is, they continue to
possess the desired biological activity of the native protein. Such
variants may result from, for example, genetic polymorphism or from
human manipulation. Variants of an IRC marker polypeptide will have
at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%,
preferably about 90% to 95% or more, and more preferably about 98%
or more sequence similarity with the amino acid sequence for a
reference IRC polypeptide as determined by sequence alignment
programs described elsewhere herein using default parameters. A
variant of an IRC polypeptide of the invention may differ from that
protein generally by as much 200, 100, 50 or 20 amino acid residues
or suitably by as few as 1-15 amino acid residues, as few as 1-10,
such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid
residue.
[0133] Variant IRC marker polypeptides may contain conservative
amino acid substitutions at various locations along their sequence,
as compared to a reference IRC marker amino add sequence. A
"conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, which can be generally
sub-classified as follows:
[0134] Acidic: The residue has a negative charge due to loss of H
ion at physiological pH and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having an acidic side chain
include glutamic acid and aspartic acid.
[0135] Basic: The residue has a positive charge due to association
with H ion at physiological pH or within one or two pH units
thereof (e.g., histidine) and the residue is attracted by aqueous
solution so as to seek the surface positions in the conformation of
a peptide in which it is contained when the peptide is in aqueous
medium at physiological pH. Amino acids having a basic side chain
include arginine, lysine and histidine.
[0136] Charged: The residues are charged at physiological pH and,
therefore, include amino acids having acidic or basic side chains
(i.e., glutamic acid, aspartic acid, arginine, lysine and
histidine).
[0137] Hydrophobic: The residues are not charged at physiological
pH and the residue is repelled by aqueous solution so as to seek
the inner positions in the conformation of a peptide in which it is
contained when the peptide is in aqueous medium. Amino acids having
a hydrophobic side chain include tyrosine, valine, isoleucine,
leucine, methionine, phenylalanine and tryptophan.
[0138] Neutral/polar: The residues are not charged at physiological
pH, but the residue is not sufficiently repelled by aqueous
solutions so that it would seek inner positions in the conformation
of a peptide in which it is contained when the peptide is in
aqueous medium. Amino acids having a neutral/polar side chain
include asparagine, glutamine, cysteine, histidine, serine and
threonine.
[0139] This description also characterizes certain amino acids as
"small" since their side chains are not sufficiently large, even if
polar groups are lacking, to confer hydrophobicity. With the
exception of proline, "small" amino acids are those with four
carbons or less when at least one polar group is on the side chain
and three carbons or less when not. Amino acids having a small side
chain include glycine, serine, alanine and threonine. The
gene-encoded secondary amino acid proline is a special case due to
its known effects on the secondary conformation of peptide chains.
The structure of proline differs from all the other
naturally-occurring amino acids in that its side chain is bonded to
the nitrogen of the .alpha.-amino group, as well as the
.alpha.-carbon. Several amino acid similarity matrices (e.g.,
PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff
et al. (1978) A model of evolutionary change in proteins. Matrices
for determining distance relationships In M. O. Dayhoff, (ed.),
Atlas of protein sequence and structure, Vol. 5, pp. 345-358,
National Biomedical Research Foundation, Washington D.C.; and by
Gonnet et al., 1992, Science 256(5062): 144301445), however,
include proline in the same group as glycine, serine, alanine and
threonine. Accordingly, for the purposes of the present invention,
proline is classified as a "small" amino acid.
[0140] The degree of attraction or repulsion required for
classification as polar or nonpolar is arbitrary and, therefore,
amino acids specifically contemplated by the invention have been
classified as one or the other. Most amino acids not specifically
named can be classified on the basis of known behavior.
[0141] Amino acid residues can be further sub-classified as cyclic
or noncyclic, and aromatic or nonaromatic, self-explanatory
classifications with respect to the side-chain substituent groups
of the residues, and as small or large. The residue is considered
small if it contains a total of four carbon atoms or less,
inclusive of the carboxyl carbon, provided an additional polar
substituent is present; three or less if not. Small residues are,
of course, always nonaromatic. Dependent on their structural
properties, amino acid residues may fall in two or more classes.
For the naturally-occurring protein amino acids, sub-classification
according to the this scheme is presented in the Table 6.
[0142] Accordingly, the present invention also contemplates
variants of the reference IRC marker polypeptide sequences or their
fragments, wherein the variants are distinguished from the
reference sequence by the addition, deletion, or substitution of
one or more amino acid residues. In general, variants will display
at least about 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% similarity to a reference IRC marker polypeptide sequence as,
for example, set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,
82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,
112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,
138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162,
164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,
190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,
216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,
242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266,
268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,
294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318,
320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344,
346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370,
372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396,
398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422,
424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448,
450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474,
476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500,
502, 504, 506, 508, 510, 512, 514 or 516. Desirably, variants will
have at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98,
99% sequence identity to a reference IRC marker polypeptide
sequence as, for example, set forth in any one of SEQ ID NO: 2, 4,
6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,
40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72,
74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,
106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,
132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,
158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182,
184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260,
262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,
288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312,
314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338,
340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364,
366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390,
392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416,
418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442,
444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,
470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494,
496, 498, 500, 502, 504, 506, 508, 510, 512, 514 or 516. Moreover,
sequences differing from the native or reference sequences by the
addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80,
90, 100, 150, 200, 300, 500 or more amino acids but which comprise
an amino acid sequence encoded by a condition-separating exon as
defined herein, are contemplated. IRC marker polypeptides also
include polypeptides that are encoded by polynucleotides that
hybridize under stringency conditions as defined herein, especially
high stringency conditions, to the IRC marker polynucleotide
sequences of the invention, or to the non-coding strand thereof, as
described above, which comprise condition-separating exons.
[0143] In some embodiments, variant polypeptides differ from an IRC
marker sequence by at least one but by less than 50, 40, 30, 20,
15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other
embodiments, variant polypeptides differ from the corresponding
sequence in any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,
88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,
220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,
272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,
324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,
350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,
376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,
402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,
454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,
480, 482, 484, 486, 488, 490; 492, 494, 496, 498, 500, 502, 504,
506, 508, 510, 512, 514 or 516 by at least 1% but less than 20%,
15%, 10% or 5% of the residues. (If this comparison requires
alignment the sequences should be aligned for maximum
similarity.
[0144] In other embodiments, a variant IRC polypeptide includes an
amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more
similarity to a corresponding sequence of an IRC marker polypeptide
as, for example, set forth in any one of SEQ ID NO: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,
80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,
110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,
136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,
162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,
188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,
214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238,
240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,
266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290,
292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316,
318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342,
344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368,
370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394,
396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420,
422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446,
448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472,
474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,
500, 502, 504, 506, 508, 510, 512, 514 or 516, and which comprise
an amino acid sequence encoded by a condition-separating exon.
[0145] IRC marker polypeptides of the invention may be prepared by
any suitable procedure known to those of skill in the art. For
example, the polypeptides may be prepared by a procedure including
the steps of: (a) preparing a chimeric construct comprising a
nucleotide sequence that encodes at least a portion of an IRC
marker polynucleotide and that is operably linked to a regulatory
element; (b) introducing the chimeric construct into a host cell;
(c) culturing the host cell to express the IRC marker polypeptide;
and (d) isolating the IRC marker polypeptide from the host cell. In
illustrative examples, the nucleotide sequence encodes at least a
portion of the sequence set forth in any one of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106,
108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,
134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,
160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,
186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210,
212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,
238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262,
264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288,
290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314,
316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340,
342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366,
368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392,
394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418,
420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444,
446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470,
472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496,
498, 500, 502, 504, 506, 508, 510, 512, 514 or 516, or a variant
thereof.
[0146] The chimeric construct is typically in the form of an
expression vector, which is suitably selected from self-replicating
extra-chromosomal vectors (e.g., plasmids) and vectors that
integrate into a host genome.
[0147] The regulatory element will generally be appropriate for the
host cell employed for expression of the IRC marker polynucleotide.
Numerous types of expression vectors and regulatory elements are
known in the art for a variety of host cells. Illustrative elements
of this type include, but are not restricted to, promoter sequences
(e.g., constitutive or inducible promoters which may be naturally
occurring or combine elements of more than one promoter), leader or
signal sequences, ribosomal binding sites, transcriptional start
and stop sequences, translational start and termination sequences,
and enhancer or activator sequences.
[0148] In some embodiments, the expression vector comprises a
selectable marker gene to permit the selection of transformed host
cells. Selectable marker genes are well known in the art and will
vary with the host cell employed.
[0149] The expression vector may also include a fusion partner
(typically provided by the expression vector) so that the IRC
marker polypeptide is produced as a fusion polypeptide with the
fusion partner.
[0150] The chimeric constructs of the invention are introduced into
a host by any suitable means including "transduction" and
"transfection", which are art recognized as meaning the
introduction of a nucleic acid, for example, an expression vector,
into a recipient cell by nucleic acid-mediated gene transfer.
"Transformation," however, refers to a process in which a host's
genotype is changed as a result of the cellular uptake of exogenous
DNA or RNA, and, for example, the transformed cell comprises the
expression system of the invention. There are many methods for
introducing chimeric constructs into cells. Typically, the method
employed will depend on the choice of host cell. Technology for
introduction of chimeric constructs into host cells is well known
to those of skill in the art. Four general classes of methods for
delivering nucleic acid molecules into cells have been described:
(1) chemical methods such as calcium phosphate precipitation,
polyethylene glycol (PEG)-mediated precipitation and lipofection;
(2) physical methods such as microinjection, electroporation,
acceleration methods and vacuum infiltration; (3) vector based
methods such as bacterial and viral vector-mediated transformation;
and (4) receptor-mediated. Transformation techniques that fall
within these and other classes are well known to workers in the
art, and new techniques are continually becoming known. The
particular choice of a transformation technology will be determined
by its efficiency to transform certain host species as well as the
experience and preference of the person practicing the invention
with a particular methodology of choice. It will be apparent to the
skilled person that the particular choice of a transformation
system to introduce a chimeric construct into cells is not
essential to or a limitation of the invention, provided it achieves
an acceptable level of nucleic acid transfer.
[0151] Recombinant IRC marker polypeptides may be produced by
culturing a host cell transformed with a chimeric construct. The
conditions appropriate for expression of the IRC marker
polynucleotide will vary with the choice of expression vector and
the host cell and are easily ascertained by one skilled in the art
through routine experimentation. Suitable host cells for expression
may be prokaryotic or eukaryotic. An illustrative host cell for
expression of a polypeptide of the invention is a bacterium. The
bacterium used may be Escherichia coli. Alternatively, the host
cell may be a yeast cell or an insect cell such as, for example,
SF9 cells that may be utilized with a baculovirus expression
system.
[0152] Recombinant IRC marker polypeptides or their fragments that
comprise an amino acid sequence encoded by a condition-separating
exon, as well as variants thereof, can be conveniently prepared
using standard protocols as described for example in Sambrook, et
al., (1989, supra), in particular Sections 16 and 17; Ausubel et
al., (1994, supra), in particular Chapters 10 and 16; and Coligan
et al., CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley &
Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
Alternatively, the IRC marker polypeptides may be synthesized by
chemical synthesis, e.g., using solution synthesis or solid phase
synthesis as described, for example, in Chapter 9 of Atherton and
Shephard (supra) and in Roberge et al (1995, Science 269: 202).
6. Thresholds
[0153] In some embodiments, the methods comprise comparing the
level or functional activity of individual expression products to
one or more preselected or threshold levels or functional
activities. Thresholds may be selected that provide an acceptable
ability to predict diagnosis, prognostic risk, treatment success,
etc. In illustrative examples, receiver operating characteristic
(ROC) curves are calculated by plotting the value of a variable
versus its relative frequency in two populations (called
arbitrarily, for example, "sepsis" and "inSIRS," "sepsis" and
"post-surgical inflammation," "sepsis" and "normal," "inSIRS" and
"post-surgical inflammation," "inSIRS" and "normal," "post-surgical
inflammation" and "normal," or simply "disease" and "normal" or
"low risk" and "high risk").
[0154] For any particular IRC marker expression product, a
distribution of expression product levels or functional activities
for subjects with and without a disease will likely overlap. Under
such conditions, a test does not absolutely distinguish "disease"
and "normal" with 100% accuracy, and the area of overlap indicates
where the test cannot distinguish "disease" and "normal." A
threshold is selected, above which (or below which, depending on
how an IRC marker expression product changes with the disease or
prognosis) the test is considered to be "positive" and below which
the test is considered to be "negative." The area under the ROC
curve is a measure of the probability that the perceived
measurement will allow correct identification of a condition (see,
e.g., Hanley et al., Radiology 143: 29-36 (1982). Alternatively, or
in addition, thresholds may be established by obtaining an earlier
marker gene expression product result from the same patient, to
which later results may be compared. In these embodiments, the
individual in effect acts as their own "control group." In markers
that increase with disease severity or prognostic risk, an increase
over time in the same patient can indicate a worsening of disease
or a failure of a treatment regimen, while a decrease over time can
indicate remission of disease or success of a treatment
regimen.
[0155] In certain embodiments, a panel of IRC marker expression
products is selected to distinguish any pair of groups selected
from "sepsis" and "inSIRS," "sepsis" and "post-surgical
inflammation," "sepsis" and "normal," "inSIRS" and "post-surgical
inflammation," "inSIRS" and "normal," "post-surgical inflammation"
and "normal," "disease" and "normal" or "low risk" and "high risk"
with at least about 70%, 80%, 85%, 90% or 95% sensitivity, suitably
in combination with at least about 70% 80%, 85%, 90% or 95%
specificity. In some embodiments, both the sensitivity and
specificity are at least about 75%, 80%, 85%, 90% or 95%.
[0156] In some embodiments, a positive likelihood ratio, negative
likelihood ratio, odds ratio, or hazard ratio is used as a measure
of the ability of the methods of the present invention to predict
disease, prognostic risk, or treatment outcome. In the case of a
positive likelihood ratio, a value of 1 indicates that a positive
result is equally likely among subjects in both the diseased group
(e.g., one of sepsis, inSIRS or post-surgical inflammation) and
control group (e.g., one of sepsis, inSIRS or post-surgical
inflammation, which is other than the diseased group, or normal); a
value greater than 1 indicates that a positive result is more
likely in the diseased group; and a value less than 1 indicates
that a positive result is more likely in the control group. In the
case of a negative likelihood ratio, a value of 1 indicates that a
negative result is equally likely among subjects in both groups; a
value greater than 1 indicates that a negative result is more
likely in the diseased group; and a value less than 1 indicates
that a negative result is more likely in the control group. In
certain embodiments, IRC markers and/or IRC marker panels are
selected to exhibit a positive or negative likelihood ratio of at
least about 1.5 or more or about 0.67 or less, at least about 2 or
more or about 0.5 or less, at least about 5 or more or about 0.2 or
less, at least about 10 or more or about 0.1 or less, or at least
about 20 or more or about 0.05 or less.
[0157] In the case of an odds ratio, a value of 1 indicates that a
positive result is equally likely among subjects in both the
diseased and control groups; a value greater than 1 indicates that
a positive result is more likely in the diseased group; and a value
less than 1 indicates that a positive result is more likely in the
control group. In certain embodiments, IRC markers and/or IRC
marker panels are selected to exhibit an odds ratio of at least
about 2 or more or about 0.5 or less, at least about 3 or more or
about 0.33 or less, at least about 4 or more or about 0.25 or less,
at least about 5 or more or about 0.2 or less, or at least about 10
or more or about 0.1 or less.
[0158] In the case of a hazard ratio, a value of 1 indicates that
the relative risk is equal in both the diseased and control groups;
a value greater than 1 indicates that the risk is greater in the
diseased group; and a value less than 1 indicates that the risk is
greater in the control group. In certain embodiments, IRC markers
and/or IRC marker panels are selected to exhibit a hazard ratio of
at least about 1.1 or more or about 0.91 or less, at least about
1.25 or more or about 0.8 or less, at least about 1.5 or more or
about 0.67 or less, at least about 2 or more or about 0.5 or less,
or at least about 2.5 or more or about 0.4 or less.
[0159] In some cases, multiple thresholds may be determined in
so-called "tertile," "quartile," or "quintile" analyses. In these
methods, the "diseased" and "control groups" (or "high risk" and
"low risk") groups are considered together as a single population,
and are divided into 3, 4, or 5 (or more) "bins" having equal
numbers of individuals. The boundary between two of these "bins"
may be considered "thresholds." A risk (of a particular diagnosis
or prognosis for example) can be assigned based on which "bin" a
test subject falls into.
[0160] In other embodiments, particular thresholds for the IRC
marker(s) measured are not relied upon to determine if the marker
level(s) obtained from a subject are correlated to a particular
diagnosis or prognosis, For example, a temporal change in the
marker(s) can be used to rule in or out one or more particular
diagnoses and/or prognoses. Alternatively, IRC marker(s) are
correlated to a condition, disease, prognosis, etc., by the
presence or absence of the IRC marker(s) in a particular assay
format. In the case of IRC marker panels, the present invention may
utilize an evaluation of the entire profile of IRC markers to
provide a single result value (e.g., a "panel response" value
expressed either as a numeric score or as a percentage risk). In
such embodiments, an increase, decrease, or other change (e.g.,
slope over time) in a certain subset of IRC markers may be
sufficient to indicate a particular condition or future outcome in
one patient, while an increase, decrease, or other change in a
different subset of IRC markers may be sufficient to indicate the
same or a different condition or outcome in another patient.
7. Methods of Detecting Aberrant IRC Marker Gene Expression
[0161] The present invention is predicated in part on the discovery
that subjects with clinical evidence of sepsis, inSIRS and
post-surgical inflammation have aberrant expression of certain
genes (referred to herein as "IRC marker genes") whose transcripts
include, but are not limited to: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,
83, 85, 87, 89, 91, 93, 95, 97, 99; 101, 103, 105, 107, 109, 111,
113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,
139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,
165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189,
191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,
217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,
243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,
269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293,
295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319,
321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345,
347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371,
373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397,
399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423,
425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449,
451, 453, 455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475,
477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501,
503, 505, 507, 509, 511, 513 or 515, as compared to one or more
control subjects selected from normal (i.e., healthy) subjects,
sepsis-negative subjects, inSIRS-negative subjects, post surgical
inflammation-negative subjects, sepsis-negative, inSIRS-negative
subjects, sepsis-negative, post surgical inflammation-negative
subjects, inSIRS-negative, post surgical inflammation-negative
subjects, sepsis-positive subjects, inSIRS-positive subjects and
post-surgical inflammation-positive subject. In some embodiments,
at least two subjects forming a control or reference population are
used for the comparison. For example, the control or reference
populations may be chosen from individuals who do not have
post-surgical inflammation ("post-surgical inflammation-negative"),
from individuals who do not have inSIRS ("inSIRS-negative"), from
individuals who do not have inSIRS but who are suffering from an
infectious process, from individuals who are suffering from
post-surgical inflammation without the presence of inSIRS or sepsis
("post-surgical inflammation-positive"), from individuals who are
suffering from inSIRS without the presence of sepsis
("inSIRS-positive"), from individuals who are suffering from the
onset of sepsis, from individuals who are sepsis-positive and
suffering from one of the stages in the progression of sepsis, or
from individuals with a physiological trauma that increases the
risk of developing sepsis. The control or reference populations may
be post-surgical inflammation-positive and are subsequently
diagnosed with inSIRS using conventional techniques. For example, a
population of post-surgical inflammation-positive patients used to
generate the reference profile may be diagnosed with inSIRS about
24, 48, 72, 96 or more hours after biological samples are taken
from them for the purposes of generating a reference IRC marker
profile. In some embodiments, the population of post-surgical
inflammation-positive individuals is diagnosed with inSIRS using
conventional techniques about 0-36 hours, about 36-60 hours, about
60-84 hours, or about 84-108 hours after the biological samples are
taken. If the marker profile is indicative of inSIRS or one of its
stages of progression, a clinician may begin treatment prior to the
manifestation of clinical symptoms.
[0162] In other embodiments, the control or reference populations
may be inSIRS-positive and are subsequently diagnosed with sepsis
using convention techniques. For example, a population of
inSIRS-positive patients used to generate the reference profile may
be diagnosed with sepsis about 24, 48, 72, 96 or more hours after
biological samples are taken from them for the purposes of
generating a reference IRC marker profile. In some embodiments, the
population of inSIRS-positive individuals is diagnosed with sepsis
using conventional techniques about 0-36 hours, about 36-60 hours,
about 60-84 hours, or about 84-108 hours after the biological
samples are taken. If the marker profile is indicative of sepsis or
one of its stages of progression, a clinician may begin treatment
prior to the manifestation of clinical symptoms of sepsis.
Treatment typically will involve examining the patient to determine
the source of the infection. Once locating the source, the
clinician typically will obtain cultures from the site of the
infection, suitably before beginning relevant empirical
antimicrobial therapy and perhaps additional adjunctive therapeutic
measures, such as draining an abscess or removing an infected
catheter.
[0163] In accordance with the present invention, comparing the
level of at one IRC marker expression product in a subject to the
level of a corresponding IRC marker expression product in a control
subject selected for example from a normal subject, a
sepsis-positive subject, an inSIRS-positive subjects and a
post-surgical inflammation-positive subject indicates whether the
subject under test is normal or has or is at risk of developing
post-surgical inflammation, inSIRS or sepsis.
[0164] Accordingly, in certain embodiments, the invention features
a method for diagnosing the presence or absence of a plurality of
conditions selected from post-surgical inflammation, inSIRS or
sepsis, or for distinguishing between those conditions in a subject
by detecting differential expression of an IRC marker expression
product between a test subject and a control subject. Accordingly,
in order to make such diagnoses, it is desirable to qualitatively
or quantitatively determine the levels of IRC marker transcripts or
the level or functional activity of IRC marker polypeptides. In
some embodiments, the presence or absence of post-surgical
inflammation, inSIRS or sepsis, or differentiation between
post-surgical inflammation, inSIRS and sepsis, is determined when
an IRC marker expression product is expressed at a detectably lower
level in a biological sample obtained from the test subject than
the level at which a corresponding IRC expression product is
expressed in a reference sample obtained from a control subject. In
other embodiments, the presence or absence of post-surgical
inflammation, inSIRS or sepsis, or differentiation between
post-surgical inflammation, inSIRS and sepsis, is determined when
na IRC marker expression product is expressed at a detectably
higher level in a biological sample obtained from the test subject
than the level at which a corresponding IRC expression product is
expressed in a reference sample obtained from a control subject.
Generally, such diagnoses are made when the level or functional
activity of an IRC marker expression product in the biological
sample varies from the level or functional activity of a
corresponding IRC marker expression product in the reference sample
by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 92%, 94%,
96%, 97%, 98% or 99%, or even by at least about 99.5%, 99.9%,
99.95%, 99.99%, 99.995% or 99.999%, or even by at least about 100%,
200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. The
corresponding IRC marker expression product is generally selected
from the same IRC marker expression product that is present in the
biological sample, an IRC expression product expressed from a
variant gene (e.g., an homologous or orthologous gene) including an
allelic variant, or a splice variant or protein product thereof. In
some embodiments, the method comprises measuring the level of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23 or 24 IRC marker expression products from an IRC
multi-transcript-producing gene selected from ANKDD1A, GABRR2,
OTX1, PANX2, RHBDF2, SLAMF7, AMBRA1, CES2, CLPB, HIPK2, C1ORF91,
NDST1, SLC36A1, ADAM19, CUL7, TG, PDCD1LG2, GRINL1A, MGRN1, SNTB2,
CDK5R1, GAA, KATNAL2, CEACAM4, ZNF335, ASPHD2, ACRC, BTNL8, MOV10,
MED12L, KLHL6, PDLIM5, GALNT10, SCRN1, VOPP1, FKBP9, KIF27, PIWIL4,
TEP1, GCH1, PRR11, CDH2, PPM1N, RRAS, DDOST, APH1A, TTL, TEX261,
COQ2, FCHSD1, BAK1, SLC25A25, RELT, ACP2, TBC1D2B, FANCA or
SLC39A11, either alone or in combination with as much as 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23 or 24 individual IRC marker expression products from each of
56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,
39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,
22, 21, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3
or 2 IRC multi-transcript-producing genes or from 1 other IRC
multi-transcript-producing gene.
[0165] In other embodiments, the methods comprise measuring the
level of one or more IRC marker polypeptides from at least one IRC
multi-transcript-producing gene as defined herein, either alone or
in combination with as much as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or
12 individual IRC marker polypeptides expressed from 56, 55, 54,
53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37,
36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1
other IRC multi-transcript-producing gene(s).
[0166] Generally, the biological sample contains blood, especially
peripheral blood, or a fraction or extract thereof. Typically, the
biological sample comprises blood cells such as mature, immature
and developing leukocytes, including lymphocytes, polymorphonuclear
leukocytes, neutrophils, monocytes, reticulocytes, basophils,
coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages,
dendritic cells natural killer cells, or fraction of such cells
(e.g., a nucleic acid or protein fraction). In specific
embodiments, the biological sample comprises leukocytes including
peripheral blood mononuclear cells (PBMC).
[0167] 7.1 Nucleic Acid-Based Diagnostics
[0168] Nucleic acid used in polynucleotide-based assays can be
isolated from cells contained in the biological sample, according
to standard methodologies (Sambrook, et al., 1989, supra; and
Ausubel et al., 1994, supra). The nucleic acid is typically
fractionated (e.g., poly A.sup.+ RNA) or whole cell RNA. Where RNA
is used as the subject of detection, it may be desired to convert
the RNA to a complementary DNA. In some embodiments, the nucleic
acid is amplified by a template-dependent nucleic acid
amplification technique. A number of template dependent processes
are available to amplify the IRC marker sequences present in a
given template sample. An exemplary nucleic acid amplification
technique is the polymerase chain reaction (referred to as PCR)
which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202
and 4,800,159, Ausubel et al. (supra), and in Innis et al., ("PCR
Protocols", Academic Press, Inc., San Diego Calif., 1990). Briefly,
in PCR, two primer sequences are prepared that are complementary to
regions on opposite complementary strands of the marker sequence.
An excess of deoxynucleoside triphosphates are added to a reaction
mixture along with a DNA polymerase, e.g., Taq polymerase. If a
cognate IRC marker sequence is present in a sample, the primers
will bind to the marker and the polymerase will cause the primers
to be extended along the marker sequence by adding on nucleotides.
By raising and lowering the temperature of the reaction mixture,
the extended primers will dissociate from the marker to form
reaction products, excess primers will bind to the marker and to
the reaction products and the process is repeated. A reverse
transcriptase PCR amplification procedure may be performed in order
to quantify the amount of mRNA amplified. Methods of reverse
transcribing RNA into cDNA are well known and described in Sambrook
et al., 1989, supra. Alternative methods for reverse transcription
utilize thermostable, RNA-dependent DNA polymerases. These methods
are described in WO 90/07641. Polymerase chain reaction
methodologies are well known in the art.
[0169] In certain advantageous embodiments, the template-dependent
amplification involves quantification of transcripts in real-time.
For example, RNA or DNA may be quantified using the Real-Time PCR
technique (Higuchi, 1992, et al., Biotechnology 10: 413-417). By
determining the concentration of the amplified products of the
target DNA in PCR reactions that have completed the same number of
cycles and are in their linear ranges, it is possible to determine
the relative concentrations of the specific target sequence in the
original DNA mixture. If the DNA mixtures are cDNAs synthesized
from RNAs isolated from different tissues or cells, the relative
abundance of the specific mRNA from which the target sequence was
derived can be determined for the respective tissues or cells. This
direct proportionality between the concentration of the PCR
products and the relative mRNA abundance is only true in the linear
range of the PCR reaction. The final concentration of the target
DNA in the plateau portion of the curve is determined by the
availability of reagents in the reaction mix and is independent of
the original concentration of target DNA. In specific embodiments,
multiplexed, tandem PCR (MT-PCR) is employed, which uses a two-step
process for gene expression profiling from small quantities of RNA
or DNA, as described for example in US Pat. Appl. Pub. No.
20070190540. In the first step, RNA is converted into cDNA and
amplified using multiplexed gene specific primers. In the second
step each individual gene is quantitated by real time PCR.
[0170] Another method for amplification is the ligase chain
reaction ("LCR"), disclosed in EPO No. 320 308. In LCR, two
complementary probe pairs are prepared, and in the presence of the
target sequence, each pair will bind to opposite complementary
strands of the target such that they abut. In the presence of a
ligase, the two probe pairs will link to form a single unit. By
temperature cycling, as in PCR, bound ligated units dissociate from
the target and then serve as "target sequences" for ligation of
excess probe pairs. U.S. Pat. No. 4,883,750 describes a method
similar to LCR for binding probe pairs to a target sequence.
[0171] Q.beta. Replicase, described in PCT Application No.
PCT/US87/00880, may also be used. In this method, a replicative
sequence of RNA that has a region complementary to that of a target
is added to a sample in the presence of an RNA polymerase. The
polymerase will copy the replicative sequence that can then be
detected.
[0172] An isothermal amplification method, in which restriction
endonucleases and ligases are used to achieve the amplification of
target molecules that contain nucleotide
5'.alpha.-thio-triphosphates in one strand of a restriction site
may also be useful in the amplification of nucleic acids in the
present invention, Walker et al., (1992, Proc. Natl. Acad. Sci.
U.S.A 89: 392-396).
[0173] Strand Displacement Amplification (SDA) is another method of
carrying out isothermal amplification of nucleic acids which
involves multiple rounds of strand displacement and synthesis,
i.e., nick translation. A similar method, called Repair Chain
Reaction (RCR), involves annealing several probes throughout a
region targeted for amplification, followed by a repair reaction in
which only two of the four bases are present. The other two bases
can be added as biotinylated derivatives for easy detection. A
similar approach is used in SDA. Target specific sequences can also
be detected using a cyclic probe reaction (CPR). In CPR, a probe
having 3' and 5' sequences of non-specific DNA and a middle
sequence of specific RNA is hybridized to DNA that is present in a
sample. Upon hybridization, the reaction is treated with RNase H,
and the products of the probe identified as distinctive products
that are released after digestion. The original template is
annealed to another cycling probe and the reaction is repeated.
[0174] Still another amplification method described in GB
Application No. 2 202 328, and in PCT Application No.
PCT/US89/01025, may be used. In the former application, "modified"
primers are used in a PCR-like, template- and enzyme-dependent
synthesis. The primers may be modified by labeling with a capture
moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In
the latter application, an excess of labeled probes are added to a
sample. In the presence of the target sequence, the probe binds and
is cleaved catalytically. After cleavage, the target sequence is
released intact to be bound by excess probe. Cleavage of the
labelled probe signals the presence of the target sequence.
[0175] Other nucleic acid amplification procedures include
transcription-based amplification systems (TAS), including nucleic
acid sequence based amplification (NASBA) and 3SR (Kwoh et al.,
1989, Proc. Natl. Acad. Sci. U.S.A., 86: 1173; Gingeras et al., PCT
Application WO 88/10315). In NASBA, the nucleic acids can be
prepared for amplification by standard phenol/chloroform
extraction, heat denaturation of a clinical sample, treatment with
lysis buffer and minispin columns for isolation of DNA and RNA or
guanidinium chloride extraction of RNA. These amplification
techniques involve annealing a primer which has target specific
sequences. Following polymerization, DNA/RNA hybrids are digested
with RNase H while double stranded DNA molecules are heat denatured
again. In either case the single stranded DNA is made fully double
stranded by addition of second target specific primer, followed by
polymerization. The double-stranded DNA molecules are then multiply
transcribed by an RNA polymerase such as T7 or SP6. In an
isothermal cyclic reaction, the RNAs are reverse transcribed into
single stranded DNA, which is then converted to double stranded
DNA, and then transcribed once again with an RNA polymerase such as
T7 or SP6. The resulting products, whether truncated or complete,
indicate target specific sequences.
[0176] Vincent and Kong disclose a method termed helicase-dependent
isothermal DNA amplification (HDA) (Vincent and Kong, EMBO Reports,
5(8):795-800, 2004). This method uses DNA helicase to separate DNA
strands and hence does not require thermal cycling. The entire
reaction can be carried out at one temperature and this method
should have broad application to point-of-care DNA diagnostics.
[0177] Davey et al., EPO No. 329 822 disclose a nucleic acid
amplification process involving cyclically synthesizing
single-stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA
(dsDNA), which may be used in accordance with the present
invention. The ssRNA is a template for a first primer
oligonucleotide, which is elongated by reverse transcriptase
(RNA-dependent DNA polymerase). The RNA is then removed from the
resulting DNA:RNA duplex by the action of ribonuclease H(RNase H,
an RNase specific for RNA in duplex with either DNA or RNA). The
resultant ssDNA is a template for a second primer, which also
includes the sequences of an RNA polymerase promoter (exemplified
by T7 RNA polymerase) 5' to its homology to the template. This
primer is then extended by DNA polymerase (exemplified by the large
"Klenow" fragment of E. coli DNA polymerase I), resulting in a
double-stranded DNA ("dsDNA") molecule, having a sequence identical
to that of the original RNA between the primers and having
additionally, at one end, a promoter sequence. This promoter
sequence can be used by the appropriate RNA polymerase to make many
RNA copies of the DNA. These copies can then re-enter the cycle
leading to very swift amplification. With proper choice of enzymes,
this amplification can be done isothermally without addition of
enzymes at each cycle. Because of the cyclical nature of this
process, the starting sequence can be chosen to be in the form of
either DNA or RNA.
[0178] Miller et al. in PCT Application WO 89/06700 disclose a
nucleic acid sequence amplification scheme based on the
hybridization of a promoter/primer sequence to a target
single-stranded DNA ("ssDNA") followed by transcription of many RNA
copies of the sequence. This scheme is not cyclic, i.e., new
templates are not produced from the resultant RNA transcripts.
Other amplification methods include "RACE" and "one-sided PCR"
(Frohman, M. A., In: "PCR Protocols: A Guide to Methods and
Applications", Academic Press, N.Y., 1990; Ohara et al., 1989,
Proc. Natl. Acad. Sci. U.S.A., 86: 5673-567).
[0179] Methods based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, may also be used for amplifying target nucleic
acid sequences. Wu et al., (1989, Genomics 4: 560).
[0180] Depending on the format, the IRC marker nucleic acid of
interest is identified in the sample directly using a
template-dependent amplification as described, for example, above,
or with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of radiolabel or fluorescent label or even
via a system using electrical or thermal impulse signals (Affymax
Technology; Bellus, 1994, J Macromol. Sci. Pure, Appl. Chem.,
A31(1): 1355-1376).
[0181] In some embodiments, amplification products or "amplicons"
are visualized in order to confirm amplification of the IRC marker
sequences. One typical visualization method involves staining of a
gel with ethidium bromide and visualization under UV light.
Alternatively, if the amplification products are integrally labeled
with radio- or fluorometrically-labelled nucleotides, the
amplification products can then be exposed to x-ray film or
visualized under the appropriate stimulating spectra, following
separation. In some embodiments, visualization is achieved
indirectly. Following separation of amplification products, a
labeled nucleic acid probe is brought into contact with the
amplified IRC marker sequence. The probe is suitably conjugated to
a chromophore but may be radiolabeled. Alternatively, the probe is
conjugated to a binding partner, such as an antigen-binding
molecule, or biotin, and the other member of the binding pair
carries a detectable moiety or reporter molecule. The techniques
involved are well known to those of skill in the art and can be
found in many standard texts on molecular protocols (e.g., see
Sambrook et al., 1989, supra and Ausubel et al. 1994, supra). For
example, chromophore or radiolabel probes or primers identify the
target during or following amplification.
[0182] In certain embodiments, target nucleic acids are quantified
using blotting techniques, which are well known to those of skill
in the art. Southern blotting involves the use of DNA as a target,
whereas Northern blotting involves the use of RNA as a target. Each
provide different types of information, although cDNA blotting is
analogous, in many aspects, to blotting or RNA species. Briefly, a
probe is used to target a DNA or RNA species that has been
immobilized on a suitable matrix, often a filter of nitrocellulose.
The different species should be spatially separated to facilitate
analysis. This often is accomplished by gel electrophoresis of
nucleic acid species followed by "blotting" on to the filter.
Subsequently, the blotted target is incubated with a probe (usually
labeled) under conditions that promote denaturation and
rehybridization. Because the probe is designed to base pair with
the target, the probe will bind a portion of the target sequence
under renaturing conditions. Unbound probe is then removed, and
detection is accomplished as described above.
[0183] Following detection/quantification, one may compare the
results seen in a given subject with a control reaction or a
statistically significant reference group or population of control
subjects as defined herein. In this way, it is possible to
correlate the amount of a IRC marker nucleic acid detected with the
progression or severity of the disease.
[0184] Also contemplated are genotyping methods and allelic
discrimination methods and technologies such as those described by
Kristensen et al. (Biotechniques 30(2): 318-322), including the use
of single nucleotide polymorphism analysis, high performance liquid
chromatography, TaqMan.RTM., liquid chromatography, and mass
spectrometry.
[0185] Also contemplated are biochip-based technologies such as
those described by Hacia et al. (1996, Nature Genetics 14: 441-447)
and Shoemaker et al. (1996, Nature Genetics 14: 450-456). Briefly,
these techniques involve quantitative methods for analysing large
numbers of genes rapidly and accurately. By tagging genes with
oligonucleotides or using fixed probe arrays, one can employ
biochip technology to segregate target molecules as high density
arrays and screen these molecules on the basis of hybridization.
See also Pease et al. (1994, Proc. Natl. Acad. Sci. U.S.A. 91:
5022-5026); Fodor et al. (1991, Science 251: 767-773). Briefly,
nucleic acid probes to IRC marker polynucleotides are made and
attached to biochips to be used in screening and diagnostic
methods, as outlined herein. The nucleic acid probes attached to
the biochip are designed to be substantially complementary to
specific expressed IRC marker nucleic acids, i.e., the target
sequence (either the target sequence of the sample or to other
probe sequences, for example in sandwich assays), such that
hybridization of the target sequence and the probes of the present
invention occurs. This complementarity need not be perfect; there
may be any number of base pair mismatches which will interfere with
hybridization between the target sequence and the nucleic acid
probes of the present invention. However, if the number of
mismatches is so great that no hybridization can occur under even
the least stringent of hybridization conditions, the sequence is
not a complementary target sequence. In certain embodiments, more
than one probe per sequence is used, with either overlapping probes
or probes to different sections of the target being used. That is,
two, three, four or more probes, with three being desirable, are
used to build in a redundancy for a particular target. The probes
can be overlapping (i.e. have some sequence in common), or
separate.
[0186] As will be appreciated by those of ordinary skill in the
art, nucleic acids can be attached to or immobilized on a solid
support in a wide variety of ways. By "immobilized" and grammatical
equivalents herein is meant the association or binding between the
nucleic acid probe and the solid support is sufficient to be stable
under the conditions of binding, washing, analysis, and removal as
outlined below. The binding can be covalent or non-covalent. By
"non-covalent binding" and grammatical equivalents herein is meant
one or more of either electrostatic, hydrophilic, and hydrophobic
interactions. Included in non-covalent binding is the covalent
attachment of a molecule, such as, streptavidin to the support and
the non-covalent binding of the biotinylated probe to the
streptavidin. By "covalent binding" and grammatical equivalents
herein is meant that the two moieties, the solid support and the
probe, are attached by at least one bond, including sigma bonds, pi
bonds and coordination bonds. Covalent bonds can be formed directly
between the probe and the solid support or can be formed by a cross
linker or by inclusion of a specific reactive group on either the
solid support or the probe or both molecules. Immobilization may
also involve a combination of covalent and non-covalent
interactions.
[0187] In general, the probes are attached to the biochip in a wide
variety of ways, as will be appreciated by those in the art. As
described herein, the nucleic acids can either be synthesized
first, with subsequent attachment to the biochip, or can be
directly synthesized on the biochip.
[0188] The biochip comprises a suitable solid or semi-solid
substrate or solid support. By "substrate" or "solid support" is
meant any material that can be modified to contain discrete
individual sites appropriate for the attachment or association of
the nucleic acid probes and is amenable to at least one detection
method. As will be appreciated by practitioners in the art, the
number of possible substrates are very large, and include, but are
not limited to, glass and modified or functionalised glass,
plastics (including acrylics, polystyrene and copolymers of styrene
and other materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon.TM., etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses,
plastics, etc. In general, the substrates allow optical detection
and do not appreciably fluorescese.
[0189] Generally the substrate is planar, although as will be
appreciated by those of skill in the art, other configurations of
substrates may be used as well. For example, the probes may be
placed on the inside surface of a tube, for flow-through sample
analysis to minimize sample volume. Similarly, the substrate may be
flexible, such as a flexible foam, including closed cell foams made
of particular plastics.
[0190] In certain embodiments, oligonucleotides probes are
synthesized on the substrate, as is known in the art. For example,
photoactivation techniques utilizing photopolymerisation compounds
and techniques can be used. In an illustrative example, the nucleic
acids are synthesized in situ, using well known photolithographic
techniques, such as those described in WO 95/25116; WO 95/35505;
U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited
within; these methods of attachment form the basis of the
Affymetrix GeneChip.TM. technology.
[0191] In an illustrative biochip analysis, oligonucleotide probes
on the biochip are exposed to or contacted with a nucleic acid
sample suspected of containing one or more IRC marker
polynucleotides under conditions favoring specific hybridization.
Sample extracts of DNA or RNA, either single or double-stranded,
may be prepared from fluid suspensions of biological materials, or
by grinding biological materials, or following a cell lysis step
which includes, but is not limited to, lysis effected by treatment
with SDS (or other detergents), osmotic shock, guanidinium
isothiocyanate and lysozyme. Suitable DNA, which may be used in the
method of the invention, includes cDNA. Such DNA may be prepared by
any one of a number of commonly used protocols as for example
described in Ausubel, et al., 1994, supra, and Sambrook, et al., et
al., 1989, supra.
[0192] Suitable RNA, which may be used in the method of the
invention, includes messenger RNA, complementary RNA transcribed
from DNA (cRNA) or genomic or subgenomic RNA. Such RNA may be
prepared using standard protocols as for example described in the
relevant sections of Ausubel, et al. 1994, supra and Sambrook, et
al. 1989, supra).
[0193] cDNA may be fragmented, for example, by sonication or by
treatment with restriction endonucleases. Suitably, cDNA is
fragmented such that resultant DNA fragments are of a length
greater than the length of the immobilized oligonucleotide probe(s)
but small enough to allow rapid access thereto under suitable
hybridization conditions. Alternatively, fragments of cDNA may be
selected and amplified using a suitable nucleotide amplification
technique, as described for example above, involving appropriate
random or specific primers.
[0194] Usually the target IRC marker polynucleotides are detectably
labeled so that their hybridization to individual probes can be
determined. The target polynucleotides are typically detectably
labeled with a reporter molecule illustrative examples of which
include chromogens, catalysts, enzymes, fluorochromes,
chemiluminescent molecules, bioluminescent molecules, lanthanide
ions (e.g., Eu.sup.34), a radioisotope and a direct visual label.
In the case of a direct visual label, use may be made of a
colloidal metallic or non-metallic particle, a dye particle, an
enzyme or a substrate, an organic polymer, a latex particle, a
liposome, or other vesicle containing a signal producing substance
and the like. Illustrative labels of this type include large
colloids, for example, metal colloids such as those from gold,
selenium, silver, tin and titanium oxide. In some embodiments in
which an enzyme is used as a direct visual label, biotinylated
bases are incorporated into a target polynucleotide. Hybridization
is detected by incubation with streptavidin-reporter molecules.
[0195] Suitable fluorochromes include, but are not limited to,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other
exemplary fluorochromes include those discussed by Dower et al.
(International Publication WO 93/06121). Reference also may be made
to the fluorochromes described in U.S. Pat. Nos. 5,573,909 (Singer
et al), 5,326,692 (Brinkley et al). Alternatively, reference may be
made to the fluorochromes described in U.S. Pat. Nos. 5,227,487,
5,274,113, 5,405,975, 5,433,896, 5,442,045, 5,451,663, 5,453,517,
5,459,276, 5,516,864, 5,648,270 and 5,723,218. Commercially
available fluorescent labels include, for example, fluorescein
phosphoramidites such as Fluoreprime.TM. (Pharmacia),
Fluoredite.TM. (Millipore) and FAM (Applied Biosystems
International)
[0196] Radioactive reporter molecules include, for example,
.sup.32P, which can be detected by an X-ray or phosphoimager
techniques.
[0197] The hybrid-forming step can be performed under suitable
conditions for hybridizing oligonucleotide probes to test nucleic
acid including DNA or RNA. In this regard, reference may be made,
for example, to NUCLEIC ACID HYBRIDIZATION, A PRACTICAL APPROACH
(Homes and Higgins, eds.) (IRL press, Washington D.C., 1985). In
general, whether hybridization takes place is influenced by the
length of the oligonucleotide probe and the polynucleotide sequence
under test, the pH, the temperature, the concentration of mono- and
divalent cations, the proportion of G and C nucleotides in the
hybrid-forming region, the viscosity of the medium and the possible
presence of denaturants. Such variables also influence the time
required for hybridization. The preferred conditions will therefore
depend upon the particular application. Such empirical conditions,
however, can be routinely determined without undue
experimentation.
[0198] In certain advantageous embodiments, high discrimination
hybridization conditions are used. For example, reference may be
made to Wallace et al. (1979, Nucl. Acids Res. 6: 3543) who
describe conditions that differentiate the hybridization of 11 to
17 base long oligonucleotide probes that match perfectly and are
completely homologous to a target sequence as compared to similar
oligonucleotide probes that contain a single internal base pair
mismatch. Reference also may be made to Wood et al. (1985, Proc.
Natl. Acid. Sci. USA 82: 1585) who describe conditions for
hybridization of 11 to 20 base long oligonucleotides using 3M
tetramethyl ammonium chloride wherein the melting point of the
hybrid depends only on the length of the oligonucleotide probe,
regardless of its GC content. In addition, Drmanac et al. (supra)
describe hybridization conditions that allow stringent
hybridization of 6-10 nucleotide long oligomers, and similar
conditions may be obtained most readily by using nucleotide
analogues such as `locked nucleic acids (Christensen et al., 2001,
Biochem J 354: 481-4).
[0199] Generally, a hybridization reaction can be performed in the
presence of a hybridization buffer that optionally includes a
hybridization-optimizing agent, such as an isostabilising agent, a
denaturing agent and/or a renaturation accelerant. Examples of
isostabilising agents include, but are not restricted to, betaines
and lower tetraalkyl ammonium salts. Denaturing agents are
compositions that lower the melting temperature of double stranded
nucleic acid molecules by interfering with hydrogen bonding between
bases in a double stranded nucleic acid or the hydration of nucleic
acid molecules. Denaturing agents include, but are not restricted
to, formamide, formaldehyde, dimethylsulfoxide, tetraethyl acetate,
urea, guanidium isothiocyanate, glycerol and chaotropic salts.
Hybridization accelerants include heterogeneous nuclear
ribonucleoprotein (hnRP) A1 and cationic detergents such as
cetyltrimethylammonium bromide (CTAB) and dodecyl trimethylammonium
bromide (DTAB), polylysine, spermine, spermidine, single stranded
binding protein (SSB), phage T4 gene 32 protein and a mixture of
ammonium acetate and ethanol. Hybridization buffers may include
target polynucleotides at a concentration between about 0.005 nM
and about 50 nM, preferably between about 0.5 nM and 5 nM, more
preferably between about 1 nM and 2 nM.
[0200] A hybridization mixture containing the target IRC marker
polynucleotides is placed in contact with the array of probes and
incubated at a temperature and for a time appropriate to permit
hybridization between the target sequences in the target
polynucleotides and any complementary probes. Contact can take
place in any suitable container, for example, a dish or a cell
designed to hold the solid support on which the probes are bound.
Generally, incubation will be at temperatures normally used for
hybridization of nucleic acids, for example, between about
20.degree. C. and about 75.degree. C., example, about 25.degree.
C., about 30.degree. C., about 35.degree. C., about 40.degree. C.,
about 45.degree. C., about 50.degree. C., about 55.degree. C.,
about 60.degree. C., or about 65.degree. C. For probes longer than
14 nucleotides, 20.degree. C. to 50.degree. C. is desirable. For
shorter probes, lower temperatures are preferred. A sample of
target polynucleotides is incubated with the probes for a time
sufficient to allow the desired level of hybridization between the
target sequences in the target polynucleotides and any
complementary probes. For example, the hybridization may be carried
out at about 45.degree. C.+/-10.degree. C. in formamide for 1-2
days.
[0201] After the hybrid-forming step, the probes are washed to
remove any unbound nucleic acid with a hybridization buffer, which
can typically comprise a hybridization optimizing agent in the same
range of concentrations as for the hybridization step. This washing
step leaves only bound target polynucleotides. The probes are then
examined to identify which probes have hybridized to a target
polynucleotide.
[0202] The hybridization reactions are then detected to determine
which of the probes has hybridized to a corresponding target
sequence. Depending on the nature of the reporter molecule
associated with a target polynucleotide, a signal may be
instrumentally detected by irradiating a fluorescent label with
light and detecting fluorescence in a fluorimeter; by providing for
an enzyme system to produce a dye which could be detected using a
spectrophotometer; or detection of a dye particle or a colored
colloidal metallic or non metallic particle using a reflectometer;
in the case of using a radioactive label or chemiluminescent
molecule employing a radiation counter or autoradiography.
Accordingly, a detection means may be adapted to detect or scan
light associated with the label which light may include
fluorescent, luminescent, focussed beam or laser light. In such a
case, a charge couple device (CCD) or a photocell can be used to
scan for emission of light from a probe:target polynucleotide
hybrid from each location in the micro-array and record the data
directly in a digital computer. In some cases, electronic detection
of the signal may not be necessary. For example, with enzymatically
generated color spots associated with nucleic acid array format,
visual examination of the array will allow interpretation of the
pattern on the array. In the case of a nucleic acid array, the
detection means is suitably interfaced with pattern recognition
software to convert the pattern of signals from the array into a
plain language genetic profile. In certain embodiments,
oligonucleotide probes specific for different IRC marker
polynucleotides are in the form of a nucleic acid array and
detection of a signal generated from a reporter molecule on the
array is performed using a `chip reader`. A detection system that
can be used by a `chip reader` is described for example by Pirrung
et al (U.S. Pat. No. 5,143,854). The chip reader will typically
also incorporate some signal processing to determine whether the
signal at a particular array position or feature is a true positive
or maybe a spurious signal. Exemplary chip readers are described
for example by Fodor et al (U.S. Pat. No., 5,925,525).
Alternatively, when the array is made using a mixture of
individually addressable kinds of labeled microbeads, the reaction
may be detected using flow cytometry.
[0203] 7.2 Protein-Based Diagnostics
[0204] Consistent with the present invention, a difference in
concentration of a IRC marker protein between a test subject or
sample and a control subject or reference sample is indicative of
the presence or absence of sepsis or inSIRS or distinguishes
between sepsis and inSIRS. IRC marker protein levels in biological
samples can be assayed using any suitable method known in the art.
For example, when a IRC marker protein is an enzyme, the protein
can be quantified based upon its catalytic activity or based upon
the number of molecules of the protein contained in a sample.
Antibody-based techniques may be employed, such as, for example,
immunohistological and immunohistochemical methods for measuring
the level of a protein of interest in a tissue sample. For example,
specific recognition is provided by a primary antibody (polyclonal
or monoclonal) and a secondary detection system is used to detect
presence (or binding) of the primary antibody. Detectable labels
can be conjugated to the secondary antibody, such as a fluorescent
label, a radiolabel, or an enzyme (e.g., alkaline phosphatase,
horseradish peroxidase) which produces a quantifiable, e.g.,
colored, product. In another suitable method, the primary antibody
itself can be detectably labeled. As a result, immunohistological
labeling of a tissue section is provided. In some embodiments, a
protein extract is produced from a biological sample (e.g., tissue,
cells) for analysis. Such an extract (e.g., a detergent extract)
can be subjected to western-blot or dot/slot assay of the level of
the protein of interest, using routine immunoblotting methods
(Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; Jalkanen et
al., 1987, J. Cell. Biol. 105: 3087-3096).
[0205] Other useful antibody-based methods include immunoassays,
such as the enzyme-linked immunosorbent assay (ELISA) and the
radioimmunoassay (MA). For example, a protein-specific monoclonal
antibody, can be used both as an immunoadsorbent and as an
enzyme-labeled probe to detect and quantify a IRC marker protein of
interest. The amount of such protein present in a sample can be
calculated by reference to the amount present in a standard
preparation using a linear regression computer algorithm (see
Lacobilli et al., 1988, Breast Cancer Research and Treatment 11:
19-30). In other embodiments, two different monoclonal antibodies
to the protein of interest can be employed, one as the
immunoadsorbent and the other as an enzyme-labeled probe.
[0206] Additionally, recent developments in the field of protein
capture arrays permit the simultaneous detection and/or
quantification of a large number of proteins. For example,
low-density protein arrays on filter membranes, such as the
universal protein array system (Ge, 2000 Nucleic Acids Res.
28(2):e3) allow imaging of arrayed antigens using standard ELISA
techniques and a scanning charge-coupled device (CCD) detector.
Immuno-sensor arrays have also been developed that enable the
simultaneous detection of clinical analytes. It is now possible
using protein arrays, to profile protein expression in bodily
fluids, such as in sera of healthy or diseased subjects, as well as
in subjects pre- and post-drug treatment.
[0207] Protein capture arrays typically comprise a plurality of
protein-capture agents each of which defines a spatially distinct
feature of the array. The protein-capture agent can be any molecule
or complex of molecules which has the ability to bind a protein and
immobilize it to the site of the protein-capture agent on the
array. The protein-capture agent may be a protein whose natural
function in a cell is to specifically bind another protein, such as
an antibody or a receptor. Alternatively, the protein-capture agent
may instead be a partially or wholly synthetic or recombinant
protein which specifically binds a protein. Alternatively, the
protein-capture agent may be a protein which has been selected in
vitro from a mutagenized, randomized, or completely random and
synthetic library by its binding affinity to a specific protein or
peptide target. The selection method used may optionally have been
a display method such as ribosome display or phage display, as
known in the art. Alternatively, the protein-capture agent obtained
via in vitro selection may be a DNA or RNA aptamer which
specifically binds a protein target (see, e.g., Potyrailo et al.,
1998 Anal. Chem. 70:3419-3425; Cohen et al., 1998, Proc. Natl.
Acad. Sci. USA 95:14272-14277; Fukuda, et al., 1997 Nucleic Acids
Symp. Ser. 37:237-238; available from SomaLogic). For example,
aptamers are selected from libraries of oligonucleotides by the
Selex.TM. process and their interaction with protein can be
enhanced by covalent attachment, through incorporation of
brominated deoxyuridine and UV-activated crosslinking
(photoaptamers). Aptamers have the advantages of ease of production
by automated oligonucleotide synthesis and the stability and
robustness of DNA; universal fluorescent protein stains can be used
to detect binding. Alternatively, the in vitro selected
protein-capture agent may be a polypeptide (e.g., an antigen) (see,
e.g., Roberts and Szostak, 1997 Proc. Natl. Acad. Sci. USA
94:12297-12302).
[0208] An alternative to an array of capture molecules is one made
through `molecular imprinting` technology, in which peptides (e.g.,
from the C-terminal regions of proteins) are used as templates to
generate structurally complementary, sequence-specific cavities in
a polymerizable matrix; the cavities can then specifically capture
(denatured) proteins which have the appropriate primary amino acid
sequence (e.g., available from ProteinPrint.TM. and Aspira
Biosystems).
[0209] Exemplary protein capture arrays include arrays comprising
spatially addressed antigen-binding molecules, commonly referred to
as antibody arrays, which can facilitate extensive parallel
analysis of numerous proteins defining a proteome or subproteome.
Antibody arrays have been shown to have the required properties of
specificity and acceptable background, and some are available
commercially (e.g., BD Biosciences, Clontech, BioRad and Sigma).
Various methods for the preparation of antibody arrays have been
reported (see, e.g., Lopez et al., 2003 J. Chromatogr. B 787:19-27;
Cahill, 2000 Trends in Biotechnology 7:47-51; U.S. Pat. App. Pub.
2002/0055186; U.S. Pat. App. Pub. 2003/0003599; PCT publication WO
03/062444; PCT publication WO 03/077851; PCT publication WO
02/59601; PCT publication WO 02/39120; PCT publication WO 01/79849;
PCT publication WO 99/39210). The antigen-binding molecules of such
arrays may recognise at least a subset of proteins expressed by a
cell or population of cells, illustrative examples of which include
growth factor receptors, hormone receptors, neurotransmitter
receptors, catecholamine receptors, amino acid derivative
receptors, cytokine receptors, extracellular matrix receptors,
antibodies, lectins, cytokines, serpins, proteases, kinases,
phosphatases, ras-like GTPases, hydrolases, steroid hormone
receptors, transcription factors, heat-shock transcription factors,
DNA-binding proteins, zinc-finger proteins, leucine-zipper
proteins, homeodomain proteins, intracellular signal transduction
modulators and effectors, apoptosis-related factors, DNA synthesis
factors, DNA repair factors, DNA recombination factors,
cell-surface antigens, hepatitis C virus (HCV) proteases and HIV
proteases.
[0210] Antigen-binding molecules for antibody arrays are made
either by conventional immunization (e.g., polyclonal sera and
hybridomas), or as recombinant fragments, usually expressed in E.
coli, after selection from phage display or ribosome display
libraries (e.g., available from Cambridge Antibody Technology,
Bioinvent, Affitech and Biosite). Alternatively, `combibodies`
comprising non-covalent associations of VH and VL domains, can be
produced in a matrix format created from combinations of
diabody-producing bacterial clones (e.g., available from Domantis).
Exemplary antigen-binding molecules for use as protein-capture
agents include monoclonal antibodies, polyclonal antibodies, Fv,
Fab, Fab' and F(ab').sub.2 immunoglobulin fragments, synthetic
stabilized Fv fragments, e.g., single chain Fv fragments (scFv),
disulfide stabilized Fv fragments (dsFv), single variable region
domains (dAbs) minibodies, combibodies and multivalent antibodies
such as diabodies and multi-scFv, single domains from camelids or
engineered human equivalents.
[0211] Individual spatially distinct protein-capture agents are
typically attached to a support surface, which is generally planar
or contoured. Common physical supports include glass slides,
silicon, microwells, nitrocellulose or PVDF membranes, and magnetic
and other microbeads.
[0212] While microdrops of protein delivered onto planar surfaces
are widely used, related alternative architectures include CD
centrifugation devices based on developments in microfluidics
(e.g., available from Gyros) and specialized chip designs, such as
engineered microchannels in a plate (e.g., The Living Chip.TM.,
available from Biotrove) and tiny 3D posts on a silicon surface
(e.g., available from Zyomyx).
[0213] Particles in suspension can also be used as the basis of
arrays, providing they are coded for identification; systems
include color coding for microbeads (e.g., available from Luminex,
Bio-Rad and Nanomics Biosystems) and semiconductor nanocrystals
(e.g., QDots.TM., available from Quantum Dots), and barcoding for
beads (UltraPlex.TM., available from Smartbeads) and multimetal
microrods (Nanobarcodes.TM. particles, available from Surromed).
Beads can also be assembled into planar arrays on semiconductor
chips (e.g., available from LEAPS technology and BioArray
Solutions). Where particles are used, individual protein-capture
agents are typically attached to an individual particle to provide
the spatial definition or separation of the array. The particles
may then be assayed separately, but in parallel, in a
compartmentalized way, for example in the wells of a microtiter
plate or in separate test tubes.
[0214] In operation, a protein sample, which is optionally
fragmented to form peptide fragments (see, e.g., U.S. Pat. App.
Pub. 2002/0055186), is delivered to a protein-capture array under
conditions suitable for protein or peptide binding, and the array
is washed to remove unbound or non-specifically bound components of
the sample from the array. Next, the presence or amount of protein
or peptide bound to each feature of the array is detected using a
suitable detection system. The amount of protein bound to a feature
of the array may be determined relative to the amount of a second
protein bound to a second feature of the array. In certain
embodiments, the amount of the second protein in the sample is
already known or known to be invariant.
[0215] For analyzing differential expression of proteins between
two cells or cell populations, a protein sample of a first cell or
population of cells is delivered to the array under conditions
suitable for protein binding. In an analogous manner, a protein
sample of a second cell or population of cells to a second array,
is delivered to a second array which is identical to the first
array. Both arrays are then washed to remove unbound or
non-specifically bound components of the sample from the arrays. In
a final step, the amounts of protein remaining bound to the
features of the first array are compared to the amounts of protein
remaining bound to the corresponding features of the second array.
To determine the differential protein expression pattern of the two
cells or populations of cells, the amount of protein bound to
individual features of the first array is subtracted from the
amount of protein bound to the corresponding features of the second
array.
[0216] In an illustrative example, fluorescence labeling can be
used for detecting protein bound to the array. The same
instrumentation as used for reading DNA microarrays is applicable
to protein-capture arrays. For differential display, capture arrays
(e.g. antibody arrays) can be probed with fluorescently labeled
proteins from two different cell states, in which cell lysates are
labeled with different fluorophores (e.g., Cy-3 and Cy-5) and
mixed, such that the color acts as a readout for changes in target
abundance. Fluorescent readout sensitivity can be amplified 10-100
fold by tyramide signal amplification (TSA) (e.g., available from
PerkinElmer Lifesciences). Planar waveguide technology (e.g.,
available from Zeptosens) enables ultrasensitive fluorescence
detection, with the additional advantage of no washing procedures.
High sensitivity can also be achieved with suspension beads and
particles, using phycoerythrin as label (e.g., available from
Luminex) or the properties of semiconductor nanocrystals (e.g.,
available from Quantum Dot). Fluorescence resonance energy transfer
has been adapted to detect binding of unlabelled ligands, which may
be useful on arrays (e.g., available from Affibody). Several
alternative readouts have been developed, including adaptations of
surface plasmon resonance (e.g., available from HTS Biosystems and
Intrinsic Bioprobes), rolling circle DNA amplification (e.g.,
available from Molecular Staging), mass spectrometry (e.g.,
available from Sense Proteomic, Ciphergen, Intrinsic and
Bioprobes), resonance light scattering (e.g., available from
Genicon Sciences) and atomic force microscopy (e.g., available from
BioForce Laboratories). A microfluidics system for automated sample
incubation with arrays on glass slides and washing has been
co-developed by NextGen and Perkin Elmer Life Sciences.
[0217] In certain embodiments, the techniques used for detection of
IRC marker expression products will include internal or external
standards to permit quantitative or semi-quantitative determination
of those products, to thereby enable a valid comparison of the
level or functional activity of these expression products in a
biological sample with the corresponding expression products in a
reference sample or samples. Such standards can be determined by
the skilled practitioner using standard protocols. In specific
examples, absolute values for the level or functional activity of
individual expression products are determined.
[0218] In specific embodiments, the diagnostic methods are
implemented using a system as disclosed, for example, in
International Publication No. WO 02/090579 and in copending PCT
Application No. PCT/AU03/01517 filed Nov. 14, 2003, comprising at
least one end station coupled to a base station. The base station
is typically coupled to one or more databases comprising
predetermined data from a number of individuals representing the
level or functional activity of IRC marker expression products,
together with indications of the actual status of the individuals
(e.g., presence, absence of sepsis or inSIRS or post-surgical
inflammation) when the predetermined data was collected. In
operation, the base station is adapted to receive from the
endstation, typically via a communications network, subject data
representing a measured or normalized level or functional activity
of at least one expression product in a biological sample obtained
from a test subject and to compare the subject data to the
predetermined data stored in the database(s). Comparing the subject
and predetermined data allows the base station to determine the
status of the subject in accordance with the results of the
comparison. Thus, the base station attempts to identify individuals
having similar parameter values to the test subject and once the
status has been determined on the basis of that identification, the
base station provides an indication of the diagnosis to the end
station.
[0219] 7.3 Kits
[0220] All the essential materials and reagents required for
detecting and quantifying IRC marker expression products may be
assembled together in a kit. The kits may also optionally include
appropriate reagents for detection of labels, positive and negative
controls, washing solutions, blotting membranes, microtiter plates
dilution buffers and the like. For example, a nucleic acid-based
detection kit may include (i) an IRC marker polynucleotide (which
may be used as a positive control), (ii) a primer or probe that
specifically hybridizes to an IRC marker polynucleotide. Also
included may be enzymes suitable for amplifying nucleic acids
including various polymerases (Reverse Transcriptase, Taq,
Sequenase.TM. DNA ligase etc. depending on the nucleic acid
amplification technique employed), deoxynucleotides and buffers to
provide the necessary reaction mixture for amplification. Such kits
also generally will comprise, in suitable means, distinct
containers for each individual reagent and enzyme as well as for
each primer or probe. Alternatively, a protein-based detection kit
may include (i) an IRC marker polypeptide (which may be used as a
positive control), (ii) an antigen-binding molecule that is
immuno-interactive with an IRC marker polypeptide. The kit can also
feature various devices and reagents for performing one of the
assays described herein; and/or printed instructions for using the
kit to quantify the expression of an sepsis marker gene.
8. Methods of Treatment or Prophylaxis
[0221] The present invention also extends to the management of
post-surgical inflammation, inSIRS and sepsis, or prevention of
further progression of post-surgical inflammation, inSIRS and
sepsis, or assessment of the efficacy of therapies in subjects
following positive diagnosis for the presence of post-surgical
inflammation, inSIRS or sepsis in a subject. Post-surgical
inflammation is typically managed using intravenous fluids,
anti-inflammatories, antibiotics or immunotherapy. However, the
management of sepsis or inSIRS conditions is generally highly
intensive and can include identification and amelioration of the
underlying cause and aggressive use of therapeutic compounds such
as, vasoactive compounds, antibiotics, steroids, antibodies to
endotoxin, anti tumour necrosis factor agents, recombinant protein
C. In addition, palliative therapies as described for example in
Cohen and Glauser (1991, Lancet 338: 736-739) aimed at restoring
and protecting organ function can be used such as intravenous
fluids and oxygen and tight glycemic control. Therapies for sepsis
are reviewed in Healy (2002, Ann. Pharmacother. 36(4): 648-54) and
Brindley (2005, CJEM. 7(4): 227) and Jenkins (2006, J Hosp Med.
1(5): 285-295).
[0222] Typically, the therapeutic agents will be administered in
pharmaceutical (or veterinary) compositions together with a
pharmaceutically acceptable carrier and in an effective amount to
achieve their intended purpose. The dose of active compounds
administered to a subject should be sufficient to achieve a
beneficial response in the subject over time such as a reduction
in, or relief from, the symptoms of post-surgical inflammation,
sepsis or inSIRS. The quantity of the pharmaceutically active
compounds(s) to be administered may depend on the subject to be
treated inclusive of the age, sex, weight and general health
condition thereof. In this regard, precise amounts of the active
compound(s) for administration will depend on the judgment of the
practitioner. In determining the effective amount of the active
compound(s) to be administered in the treatment or prevention of
post-surgical inflammation, sepsis or inSIRS, the medical
practitioner or veterinarian may evaluate severity of any symptom
associated with the presence of post-surgical inflammation, sepsis
or inSIRS including, inflammation, blood pressure anomaly,
tachycardia, tachypnea fever, chills, vomiting, diarrhoea, skin
rash, headaches, confusion, muscle aches, seizures. In any event,
those of skill in the art may readily determine suitable dosages of
the therapeutic agents and suitable treatment regimens without
undue experimentation.
[0223] The therapeutic agents may be administered in concert with
adjunctive (palliative) therapies to increase oxygen supply to
major organs, increase blood flow to major organs and/or to reduce
the inflammatory response. Illustrative examples of such adjunctive
therapies include non steroidal-anti inflammatory drugs (NSAIDs),
intravenous saline and oxygen.
[0224] In order that the invention may be readily understood and
put into practical effect, particular preferred embodiments will
now be described by way of the following non-limiting examples.
EXAMPLES
Example 1
Identification of Diagnostic Genes that Distinguish Between
Post-Surgical Inflammation, Sepsis and inSIRS
Experimental Disease Trial Designs
[0225] Clinical trials were performed to determine whether
transcripts of genes could distinguish between patients with
sepsis, inSIRS and post-surgical inflammation.
[0226] Blood samples were collected at various time points to
provide time course data and gene expression was analysed using an
Affymetrix exon array (Affymetrix HuEx-1.0) Analysis of these data
(see "Identification of Diagnostic Marker Genes" below) revealed
235 specific genes that show evidence of splice variation that also
differ in expression between sepsis-positive patients,
inSIRS-positive patients and post-surgical patients. Of these 235
only a limited number (57) were identified that can be used as
classifiers to distinguish between these patient groups. The 57
genes produce 258 transcripts that are differentially expressed
between post-surgical inflammation and inSIRS, post-surgical
inflammation and sepsis and sepsis and inSIRS. It is possible to
design a nucleic acid assay that measures the RNA level in the
sample corresponding to at least one and desirably at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 64, 55,
56, 57 IRC marker transcripts, representative transcript sequences
of which are set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85,
87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,
117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,
143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167,
169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193,
195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219,
221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,
247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,
273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297,
299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323,
325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349,
351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375,
377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401,
403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427,
429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453,
455, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479,
481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505,
507, 509, 511, 513 or 515. Alternatively, or in addition, it is
possible to design an assay that measures the protein level in the
sample corresponding to at least one and desirably at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38
39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 64, 55,
56, 57 IRC marker polypeptides, representative amino acid sequences
of which are set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,
86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,
116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,
142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166,
168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192,
194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,
220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,
272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296,
298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322,
324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348,
350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374,
376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400,
402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426,
428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452,
454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478,
480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,
506, 508, 510, 512, 514 or 516.
Materials and Methods
Study Design
[0227] In Phase I of a sepsis clinical research program, the trial
was conducted at a single tertiary referral centre. Intensive care
sepsis patients, as well as healthy controls were prospectively
enrolled and attended a single visit where 5 mL of blood was
collected for gene expression analysis using an Affymetrix exon
array.
[0228] A definitive diagnosis of sepsis was unlikely to be known at
the time patients were enrolled in the Phase II study, and thus
confirmation of sepsis diagnosis and the assignment of patients to
the sepsis cohorts was made retrospectively. Data collected from
participants not diagnosed with inSIRS or sepsis were only assessed
for frequency of adverse events.
[0229] Clinical data collection and blood specimens were not
collected until after surgery during their post-operative admission
to the ICU. Patients who had clinical signs and/or symptoms of
sepsis or inSIRS were consented and enrolled into the study as soon
as possible after they have been identified, in most cases within
24 hours of admission. Final assessment of whether the participant
had inSIRS or sepsis or post-surgical inflammation was made
retrospectively as clinical information became available.
Study Population
[0230] In the Phase I clinical trial, 12 patients presenting with
clinical signs and symptoms of sepsis (SIRS criteria as well as
suspected infection) from the ICU were enrolled. A further 10 male
and 10 female control participants from within were also enrolled.
Study participants were all over 18 years and either they or their
surrogate decision maker signed and dated the clinical trial
information sheet and consent form. All of the control participants
were considered to be in good health based on an abbreviated
physical examination by the Principal Investigator, and known
medical history at the time of clinical trial enrolment.
[0231] Phase II of this clinical research program comprised of two
cohorts of patients and included a cohort of 36 inSIRS patients
with clinical signs and symptoms consistent with inSIRS, and a
cohort of 17 patients with the clinical signs and symptoms
consistent with sepsis.
[0232] Patients or their surrogate decision maker were offered the
opportunity to participate in this study if the patient presented
with signs and symptoms of either inSIRS or sepsis at the time of
admission to ICU. All inSIRS and sepsis participants displayed
clinical signs of fever, hypotension, leukocytosis or leucopoenia
and decreased peripheral vascular resistance. These criteria are
based on the American College of Physicians and the Society of
Critical Care Medicine standard definition of sepsis. That is,
inSIRS and sepsis participants needed a variable combination of
clinical conditions including two or more of the following within
the last 24 hours: temperature >38.degree. C. or <36.degree.
C.; heart rate >90 beats/min; respiratory rate >20
breathes/min or a PaCO.sub.2 of <4.3 kPa (<32 mm Hg); and
evidence of a white blood cell count <4,000 cells/mm.sup.3
(<4.times.10.sup.9 cells/L) or >12,000 cells/mm.sup.3
(>12.times.10.sup.9 cells/L) or >10% immature neutrophils
(band forms). Participants were excluded if they had any chronic
systemic immune-inflammatory disorders including SLE, Crohn's
disease, IDDM; were transplant recipients or were currently
receiving chemotherapy treatment for cancer. Most patients had
other underlying co-morbidities. Patients who were admitted for
planned major open surgery were approached and consented prior to
the surgical procedure, and their study blood samples drawn after
surgery during their post-operative admission to the ICU. All study
participants were 18 years of age or older and had a body mass
index of less than 40.
Collection of Data
[0233] Demography, vital signs measurements (blood pressure, heart
rate, respiratory rate, oxygen saturation, temperature), hematology
(full blood count), clinical chemistry. (urea, electrolytes, liver
function enzymes, blood glucose) as well as microbial status was
recorded. Blood was drawn into maximally 6 PAXgene tubes for gene
expression analysis using RT-PCR.
Blood Collection
[0234] Blood is collected for the purpose of extraction of high
quality RNA or protein. Suitable blood collection tubes for the
collection, preservation, transport and isolation of RNA include
PAXgene.TM. tubes (PreAnalytix Inc., Valencia, Calif., USA).
Alternatively, blood can be collected into tubes containing
solutions designed for the preservation of nucleic acids (available
from Roche, Ambion, Invitrogen and ABI). For the determination of
protein levels, 50 mL of blood is prevented from clotting by
collection into a tube containing 4 mL of 4% sodium citrate. White
blood cells and plasma are isolated and stored frozen for later
analysis and detection of specific proteins. PAXgene tubes can be
kept at room temperature prior to RNA extraction. Clinical signs
are recorded in a standard format.
Total RNA Extraction
[0235] A kit available from Qiagen Inc (Valencia, Calif., USA) has
the reagents and instructions for the isolation of total RNA from
2.5 mL blood collected in the PAXgene Blood RNA Tube. Isolation
begins with a centrifugation step to pellet nucleic acids in the
PAXgene blood RNA tube. The pellet is washed and resuspended and
incubated in optimized buffers together with Proteinase K to bring
about protein digestion. An additional centrifugation is carried
out to remove residual cell debris and the supernatant is
transferred to a fresh microcentrifuge tube. Ethanol is added to
adjust binding conditions, and the lysate is applied to the PAXgene
RNA spin column. During brief centrifugation, RNA is selectively
bound to the silica-gel membrane as contaminants pass through.
Remaining contaminants are removed in three efficient wash steps
and RNA is then eluted in Buffer BR5.
[0236] Determination of RNA quantity and quality is necessary prior
to proceeding and can be achieved using an Agilent Bioanalyzer and
Absorbance 260/280 ratio using a spectrophotometer.
Choice of Method
[0237] Measurement of specific RNA levels in a tissue sample can be
achieved using a variety of technologies. Two common and readily
available technologies that are well known in the art are: [0238]
GeneChip.RTM. analysis using Affymetrix technology. [0239]
Real-Time Polymerase Chain Reaction (TaqMan.TM. from Applied
Biosystems for example).
[0240] GeneChips.RTM. quantitate RNA by detection of labeled cRNA
hybridized to short oligonucleotides built on a silicon substrate.
Details on the technology and methodology can be found at
www.affymetrix.com.
[0241] Real-Time Polymerase Chain Reaction (RT-PCR) quantitates RNA
using two PCR primers, a labeled probe and a thermostable DNA
polymerase. As PCR product is generated a dye is released into
solution and detected. Internal controls such as 18S RNA probes are
often used to determine starting levels of total RNA in the sample.
Each gene and the internal control are run separately. Details on
the technology and methods can be found at www.appliedbiosytems.com
or www.qiagen.com or www.biorad.com. Applied Biosystems offer a
service whereby the customer provides DNA sequence information and
payment and is supplied in return all of the reagents required to
perform RT-PCR analysis on individual genes.
[0242] GeneChip.RTM. analysis has the advantage of being able to
analyze thousands of genes at a time. However it is expensive and
takes over 3 days to perform a single assay. RT-PCR generally only
analyses one gene at a time, but is inexpensive and can be
completed within a single day.
[0243] RT-PCR is the method, of choice for gene expression analysis
if the number of specific genes to be analyzed is less than 20.
GeneChip.RTM. or other gene expression analysis technologies (such
as Illumina Bead Arrays) are the method of choice when many genes
need to be analyzed simultaneously.
[0244] The methodology for GeneChip.RTM. data generation and
analysis and Real Time PCR is presented below in brief.
GeneChip.RTM. Data Generation
cDNA & cRNA Generation
[0245] The following method for cDNA and cRNA generation from total
RNA has been adapted from the protocol provided and recommended by
Affymetrix (www.affymetrix.com).
[0246] The steps are: [0247] A total of 3 .mu.g of total RNA is
used as a template to generate double stranded cDNA. [0248] cRNA is
generated and labeled using biotinylated Uracil (dUTP). [0249]
biotin-labeled cRNA is cleaned and the quantity determined using a
spectrophotometer and MOPS gel analysis. [0250] labeled cRNA is
fragmented to .about.300 bp in size. [0251] RNA quantity is
determined on an Agilent "Lab-on-a-Chip" system (Agilent
Technologies).
Hybridization, Washing & Staining
[0252] The steps are: [0253] A hybridization cocktail is prepared
containing 0.05 .mu.g/.mu.L of labeled and fragmented cRNA,
spike-in positive hybridization controls, and the Affymetrix
oligonucleotides B2, bioB, bioC, bioD and cre. [0254] The final
volume (80 .mu.L) of the hybridization cocktail is added to the
GeneChip.RTM. cartridge. [0255] The cartridge is placed in a
hybridization oven at constant rotation for 16 hours. [0256] The
fluid is removed from the GeneChip.RTM. and stored. [0257] The
GeneChip.RTM. is placed in the fluidics station. [0258] The
experimental conditions for each GeneChip.RTM. are recorded as an
.EXP file. [0259] All washing and staining procedures are carried
out by the Affymetrix fluidics station with an attendant providing
the appropriate solutions. [0260] The GeneChip.RTM. is washed,
stained with steptavidin-phycoerythin dye and then washed again
using low salt solutions. [0261] After the wash protocols are
completed, the dye on the probe array is `excited` by laser and the
image captured by a CCD camera using an Affymetrix Scanner
(manufactured by Agilent).
Scanning & Data File Generation
[0262] The scanner and MAS 5 software generates an image file from
a single GeneChip.RTM. called a .DAT file.
[0263] The .DAT file is then pre-processed prior to any statistical
analysis.
[0264] Data pre-processing steps (prior to any statistical
analysis) include: [0265] .DAT File Quality Control (QC). [0266]
.CEL File Generation. [0267] Scaling and Normalization.
.DAT File Quality Control
[0268] The .DAT file is an image. The image is inspected manually
for artifacts (e.g. high/low intensity spots, scratches, high
regional or overall background). (The B2 oligonucleotide
hybridization performance is easily identified by an alternating
pattern of intensities creating a border and array name.) The MAS 5
software used the B2 oligonucleotide border to align a grid over
the image so that each square of oligonucleotides was centered and
identified.
[0269] The other spiked hybridization controls (bioB, bioC, bioD
and cre) are used to evaluate sample hybridization efficiency by
reading "present" gene detection calls with increasing signal
values, reflecting their relative concentrations. (If the .DAT file
is of suitable quality it is converted to an intensity data file
(.CEL file) by Affymetrix MAS 5 software).
.CEL File Generation
[0270] The .CEL files generated by the MAS 5 software from .DAT
files contain calculated raw intensities for the probe sets. Gene
expression data is obtained by subtracting a calculated background
from each cell value. To eliminate negative intensity values, a
noise correction fraction based from a local noise value from the
standard deviation of the lowest 2% of the background is
applied.
[0271] All .CEL files generated from the GeneChips.RTM. are
subjected to specific quality metrics parameters.
[0272] Some metrics are routinely recommended by Affymetrix and can
be determined from Affymetrix internal controls provided as part of
the GeneChip.RTM.. Other metrics are based on experience and the
processing of many GeneChips.RTM..
[0273] Analysis of GeneChip.RTM. Data
[0274] Two illustrative approaches to normalising data may be used:
[0275] Affymetrix MAS 5 Algorithm. [0276] Robust Multi-chip
Analysis (RMA) algorithm of Irizarry (Irizarray et al., 2002,
Biostatistics (in print)).
[0277] Those of skill in the art will recognize that many other
approaches might be adopted, without materially affecting the
invention.
Preprocessing
[0278] The arrays were preprocessed using the Affymetrix Power
Tools (APT) apt-probeset-summarize program. The analysis used the
array description files current at the time, (\HuEx-1
0-st-v2.r2.pgf'' and \HuEx-1 0-st-v2.r2.clf''), the antigenomic
probes for background (\HuEx-1 0-st-v2.r2.antigenomic.bgp'') and
the standard QC probes (\HuEx-1 0-st-v2.r2.qcc''). Additionally, in
all the analyses, the Robust Multichip Average (RMA) approach was
used.
[0279] Using various Affymetrix mapping files, it is possible to
compute expression measures at either the Exon or Gene level, and
for subsets of Exons or Genes entitled, Core, Extend or Full. To
date in subsequent analysis the focus has been on the Core set of
Exons and Genes as these are the most well understood and annotated
subsets. There is an exon analysis package available for the R
statistical software package (www.r-project.org) called exonmap. It
is provided by the X:Map genome browser project
(http://xmap.picr.man.ac.uk). Exonmap provides an alternative chip
description \exon.pmcdf'' that can be used to produce exon level
RMA normalised measures of expression for the Core set of exons. On
comparison with the output of the APT utilities, the differences
were found to be minor. Since the APT utilities also provide gene
level measures, these version were used throughout.
Quality Checking
[0280] The APT utility provides various quality control summaries
including the use of boxplots of the mean expression levels for the
positive and negative controls.
Model for the Data
[0281] The data were analysed to identify differential features
(exons or genes) using the linear model approach embodied in the
limma package of R. limma proceeds by estimating the coefficients
for each feature and computing a moderated t-statistic for each
contrast of interest. In addition, an overall F-statistic is
computed for the 3 contrasts together. The equivalent p-values can
then be adjusted for multiple tests in various ways. In this case,
Holm's method of adjustment and Benjamini & Hochberg's false
discovery rate (FDR).
Affymetrix MAS 5 Algorithm
[0282] .CEL files are used by Affymetrix MAS 5 software to
normalize or scale the data. Scaled data from one chip are compared
to similarly scaled data from other chips.
[0283] Affymetrix MAS 5 normalization is achieved by applying the
default "Global Scaling" option of the MAS 5 algorithm to the .CEL
files. This procedure subtracts a robust estimate of the center of
the distribution of probe values, and divides by a robust estimate
of the probe variability. This produces a set of chips with common
location and scale at the probe level.
[0284] Gene expression indices are generated by a robust averaging
procedure on all the probe pairs for a given gene. The results are
constrained to be non-negative.
[0285] Given that scaling takes place at the level of the probe,
rather than at the level of the gene, it is possible that even
after normalization there may be chip-to-chip differences in
overall gene expression level. Following standard MAS5
normalization, values for each gene were de-trended with respect to
median chip intensity. That is, values for each gene were regressed
on the median chip intensity, and residuals were calculated. These
residuals were taken as the de-trended estimates of expression for
each gene
[0286] Median chip intensity was calculated using the Affymetrix
MAS5 algorithm, but with a scale factor fixed at one.
RMA Algorithm
[0287] This algorithm quantifies the expression of a set of chips,
rather than of a single chip. It estimates background intensities
using a robust statistical model applied to perfect match probe
data. It does not make use of mis-match probe data. Following
implicit background correction, chips are processed using Quantile
Quantile normalization (Rizarray et al., 2002, Biostatistics (in
print)).
DNA Extraction
[0288] A kit available from Qiagen Inc (Valencia, Calif., USA) has
the reagents and instructions for the isolation of total DNA from
8.5 mL blood collected in the PAXgene Blood DNA Tube. Isolation
begins with the addition of additional lysis solution followed by a
centrifugation step. The pellet is washed and resuspended and
incubated in optimized buffers together with Proteinase K to bring
about protein digestion. DNA is precipitated using alcohol and an
additional centrifugation is carried out to pellet the nucleic
acid. Remaining contaminants are removed in a wash step and the DNA
is then resuspended in Buffer BG4.
[0289] Determination of DNA quantity and quality is necessary prior
to proceeding and can be achieved using a spectrophotometer or
agarose gel electrophoresis.
Genotyping Analysis
[0290] Many methods are available to genotype DNA. A review of
allelic discrimination methods can be found in Kristensen et al.
(Biotechniques 30(2): 318-322 (2001). An illustrative method for
genotyping using allele-specific PCR is described here.
Primer Design
[0291] Upstream and downstream PCR primers specific for particular
alleles can be designed using freely available computer programs,
such as Primer3
(http://frodo.wi.mit.edu/primer3/primer3_code.html). Alternatively
the DNA sequences of the various alleles can be aligned using a
program such as ClustalW (http://www.ebi.ac.uk/clustalw/) and
specific primers designed to areas where DNA sequence differences
exist but retaining enough specificity to ensure amplification of
the correct amplicon. Preferably a PCR amplicon is designed to have
a restriction enzyme site in one allele but not the other. Primers
are generally 18-25 base pairs in length with similar melting
temperatures.
PCR Amplification
[0292] The composition of PCR reactions has been described
elsewhere (Clinical Applications of PCR, Dennis Lo (Editor),
Blackwell Publishing, 1998). Briefly, a reaction contains primers,
DNA, buffers and a thermostable polymerase enzyme. The reaction is
cycled (up to 50 times) through temperature steps of denaturation,
hybridization and DNA extension on a thermocycler such as the MJ
Research Thermocycler model PTC-96V.
DNA Analysis
[0293] PCR products can be analyzed using a variety of methods
including size differentiation using mass spectrometry, capillary
gel electrophoresis and agarose gel electrophoresis. If the PCR
amplicons have been designed to contain differential restriction
enzyme sites, the DNA in the PCR reaction is purified using
DNA-binding columns or precipitation and re-suspended in water, and
then restricted using the appropriate restriction enzyme. The
restricted DNA can then be run on an agarose gel where DNA is
separated by size using electric current. Various alleles of a gene
will have different sizes depending on whether they contain
restriction sites. Thus, homozygotes and heterozygotes can be
determined.
Real-Time PCR Data Generation
[0294] Background information for conducting Real-time PCR may be
obtained, for example, at
http://dorakmt.tripod.com/genetics/realtime.html and in a review by
Bustin SA (2000, J Mol Endocrinol 25:169-193).
TaqMan.TM. Primer and Probe Design Guidelines
[0295] 1. The Primer Express.TM. (ABI) software designs primers
with a melting temperature (Tm) of 58-60.degree. C., and probes
with a Tm value of 10.degree. C. higher. The Tm of both primers
should be equal.
[0296] 2. Primers should be 15-30 bases in length.
[0297] 3. The G+C content should ideally be 30-80%. If a higher G+C
content is unavoidable, the use of high annealing and melting
temperatures, cosolvents such as glycerol, DMSO, or 7-deaza-dGTP
may be necessary:
[0298] 4. The run of an identical nucleotide should be avoided.
This is especially true for G, where runs of four or more Gs is not
allowed.
[0299] 5. The total number of Gs and Cs in the last five
nucleotides at the 3' end of the primer should not exceed two (the
newer version of the software has an option to do this
automatically). This helps to introduce relative instability to the
3' end of primers to reduce non-specific priming. The primer
conditions are the same for SYBR Green assays.
[0300] 6. Maximum amplicon size should not exceed 400 by (ideally
50-150 bases). Smaller amplicons give more consistent results
because PCR is more efficient and more tolerant of reaction
conditions (the short length requirement has nothing to do with the
efficiency of 5' nuclease activity).
[0301] 7. The probes should not have runs of identical nucleotides
(especially four or more consecutive Gs), G+C content should be
30-80%, there should be more Cs than Gs, and not a G at the 5' end.
The higher number of Cs produces a higher .alpha.Rn. The choice of
probe should be made first.
[0302] 8. To avoid false-positive results due to amplification of
contaminating genomic DNA in the cDNA preparation, it is preferable
to have primers spanning exon-exon junctions. This way, genomic DNA
will not be amplified (the PDAR kit for human GAPDH amplification
has such primers),
[0303] 9. If a TaqMan.TM. probe is designed for allelic
discrimination, the mismatching nucleotide (the polymorphic site)
should be in the middle of the probe rather than at the ends,
[0304] 10. Use primers that contain dA nucleotides near the 3' ends
so that any primer-dimer generated is efficiently degraded by
AmpErase.TM. UNG (mentioned in p. 9 of the manual for EZ RT-PCR
kit; P/N402877). If primers cannot be selected with dA nucleotides
near the ends, the use of primers with 3' terminal dU-nucleotides
should be considered.
[0305] (See also the general principles of PCR Primer Design by
InVitroGen.)
General Method
[0306] 1. Reverse transcription of total RNA to cDNA should be done
with random hexamers (not with oligo-dT). If oligo-dT has to be
used long mRNA transcripts or amplicons greater than two kilobases
upstream should be avoided, and 18S RNA cannot be used as
normalizer,
[0307] 2. Multiplex PCR will only work properly if the control
primers are limiting (ABI control reagents do not have their
primers limited),
[0308] 3. The range of target cDNA used is 10 ng to 1 .mu.g. If DNA
is used (mainly for allelic discrimination studies), the optimum
amount is 100 ng to 1 .mu.g,
[0309] 4. It is ideal to treat each RNA preparation with RNAse free
DNAse to avoid genomic DNA contamination. Even the best RNA
extraction methods yield some genomic DNA. Of course, it is ideal
to have primers not amplifying genomic DNA at all but sometimes
this may not be possible,
[0310] 5. For optimal results, the reagents (before the preparation
of the PCR mix) and the PCR mixture itself (before loading) should
be vortexed and mixed well. Otherwise there may be shifting Rn
value during the early (0-5) cycles of PCR. It is also important to
add probe to the buffer component and allow it to equilibrate at
room temperature prior to reagent mix formulation.
TaqMan.TM. Primers and Probes
[0311] The TaqMan.TM. probes ordered from ABI at midi-scale arrive
already resuspended at 100 .quadrature.M. If a 1/20 dilution is
made, this gives a 5 .mu.M solution. This stock solution should be
aliquoted, frozen and kept in the dark. Using 1 .mu.L of this in a
50 .mu.L reaction gives the recommended 100 nM final
concentration.
[0312] The primers arrive lyophilized with the amount given on the
tube in pmols (such as 150.000 pmol which is equal to 150 nmol). If
X nmol of primer is resuspended in X .mu.L of H2O, the resulting
solution is 1 mM. It is best to freeze this stock solution in
aliquots. When the 1 mM stock solution is diluted 1/100, the
resulting working solution will be 10 .mu.M. To get the recommended
50-900 nM final primer concentration in 50 .mu.L reaction volume,
0.25-4.50 .quadrature.L should be used per reaction (2.5 .mu.L for
500 nM final concentration).
[0313] The PDAR primers and probes are supplied as a mix in one
tube. They have to be used 2.5 .mu.L in a 50 .mu.L reaction
volume.
Setting Up One-Step TaqMan.TM. Reaction
[0314] One-step real-time PCR uses RNA (as opposed to cDNA) as a
template. This is the preferred method if the RNA solution has a
low concentration but only if singleplex reactions are run. The
disadvantage is that RNA carryover prevention enzyme AmpErase
cannot be used in one-step reaction format. In this method, both
reverse transcriptase and real-time PCR take place in the same
tube. The downstream PCR primer also acts as the primer for reverse
transcriptase (random hexamers or oligo-dT cannot be used for
reverse transcription in one-step RT-PCR). One-step reaction
requires higher dNTP concentration (greater than or equal to 300 mM
vs 200 mM) as it combines two reactions needing dNTPs in one. A
typical reaction mix for one-step PCR by Gold RT-PCR kit is as
follows:
TABLE-US-00001 H.sub.2O + RNA: 20.5 .mu.L [24 .mu.L if PDAR is
used] 10X TaqMan buffer: 5.0 .mu.L MgCl.sub.2 (25 mM): 11.0 .mu.L
dATP (10 mM): 1.5 .mu.L [for final concentration of 300 .mu.M] dCTP
(10 mM): 1.5 .mu.L [for final concentration of 300 .mu.M] dGTP (10
mM): 1.5 .mu.L [for final concentration of 300 .mu.M] dUTP (20 mM):
1.5 .mu.L [for final concentration of 600 .mu.M] Primer F (10
.mu.M) *: 2.5 .mu.L [for final concentration of 500 nM] Primer R
(10 .mu.M) *: 2.5 .mu.L [for final concentration of 500 nM] TaqMan
Probe *: 1.0 .mu.L [for final concentration of 100 nM] AmpliTaq
Gold: 0.25 .mu.L [can be increased for higher efficiency] Reverse
Transcriptase: 0.25 .mu.L RNAse inhibitor: 1.00 .mu.L * If a PDAR
is used, 2.5 .mu.L of primer + probe mix used.
[0315] Ideally 10 pg-100 ng RNA should be used in this reaction.
Note that decreasing the amount of template from 100 ng to 50 ng
will increase the C.sub.T value by 1. To decrease a C.sub.T value
by 3, the initial amount of template should be increased 8-fold.
ABI claims that 2 picograms of RNA can be detected by this system
and the maximum amount of RNA that can be used is 1 microgram. For
routine analysis, 10 pg-100 ng RNA and 100 pg-1 .mu.g genomic DNA
can be used.
Cycling Parameters for One-Step PCR
[0316] Reverse transcription (by MuLV) 48.degree. C. for 30
min.
[0317] AmpliTaq activation 95.degree. C. for 10 min.
[0318] PCR: denaturation 95.degree. C. for 15 sec and
annealing/extension 60.degree. C. for 1 min (repeated 40 times) (On
ABI 7700, minimum holding time is 15 seconds.)
[0319] The recently introduced EZ One-Step.TM. RT-PCR kit allows
the use of UNG as the incubation time for reverse transcription is
60.degree. C. thanks to the use of a thermostable reverse
transcriptase. This temperature also a better option to avoid
primer dimers and non-specific bindings at 48.degree. C.
Operating the ABI 7700
[0320] Make sure the following before starting a run:
[0321] 1. Cycle parameters are correct for the run.
[0322] 2. Choice of spectral compensation is correct (off for
singleplex, on for multiplex reactions).
[0323] 3. Choice of "Number of PCR Stages" is correct in the
Analysis Options box (Analysis/Options). This may have to be
manually assigned after a run if the data is absent in the
amplification plot but visible in the plate view, and the X-axis of
the amplification is displaying a range of 0-1 cycles.
[0324] 4. No Template Control is labeled as such (for accurate
.quadrature.Rn calculations).
[0325] 5. The choice of dye component should be made correctly
before data analysis.
[0326] 6. You must save the run before it starts by giving it a
name (not leaving as untitled). Also at the end of the run, first
save the data before starting to analyze.
[0327] 7. The ABI software requires extreme caution. Do not attempt
to stop a run after clicking on the Run button. You will have
problems and if you need to switch off and on the machine, you have
to wait for at least an hour to restart the run.
[0328] When analyzing the data, remember that the default setting
for baseline is 3-15. If any CT value is <15, the baseline
should be changed accordingly (the baseline stop value should be
1-2 smaller than the smallest CT value). For a useful discussion of
this matter, see the ABI Tutorial on Setting Baselines and
Thresholds. (Interestingly, this issue is best discussed in the
manual for TaqMan.TM. Human Endogenous Control Plate.)
[0329] If the results do not make sense, check the raw spectra for
a possible CDC camera saturation during the run. Saturation of CDC
camera may be prevented by using optical caps rather than optical
adhesive cover. It is also more likely to happen when SYBR Green I
is used, when multiplexing and when a high concentration of probe
is used.
Interpretation of Results
[0330] At the end of each reaction, the recorded fluorescence
intensity is used for the following calculations:
[0331] Rn+ is the Rn value of a reaction containing all components,
Rn- is the Rn value of an unreacted sample (baseline value or the
value detected in NTC). .DELTA.Rn is the difference between Rn+ and
Rn-. It is an indicator of the magnitude of the signal generated by
the PCR.
[0332] There are three illustrative methods to quantitate the
amount of template:
[0333] 1. Absolute standard method: In this method, a known amount
of standard such as in vitro translated RNA (cRNA) is used.
[0334] 2. Relative standard: Known amounts of the target nucleic
acid are included in the assay design in each run,
[0335] 3. Comparative CT method: This method uses no known amount
of standard but compares the relative amount of the target sequence
to any of the reference values chosen and the result is given as
relative to the reference value (such as the expression level of
resting lymphocytes or a standard cell line).
The Comparative CT Method (.DELTA..DELTA.CT) for Relative
Quantitation of Gene Expression
[0336] This method enables relative quantitation of template and
increases sample throughput by eliminating the need for standard
curves when looking at expression levels relative to an active
reference control (normalizer). For this method to be successful,
the dynamic range of both the target and reference should be
similar. A sensitive method to control this is to look at how
.DELTA.C.sub.T (the difference between the two C.sub.T values of
two PCRs for the same initial template amount) varies with template
dilution. If the efficiencies of the two amplicons are
approximately equal, the plot of log input amount versus
.DELTA.C.sub.T will have a nearly horizontal line (a slope of
<0.10). This means that both PCRs perform equally efficiently
across the range of initial template amounts. If the plot shows
unequal efficiency, the standard curve method should be used for
quantitation of gene expression. The dynamic range should be
determined for both (1) minimum and maximum concentrations of the
targets for which the results are accurate and (2) minimum and
maximum ratios of two gene quantities for which the results are
accurate. In conventional competitive RT-PCR, the dynamic range is
limited to a target-to-competitor ratio of about 10:1 to 1:10 (the
best accuracy is obtained for 1:1 ratio). The real-time PCR is able
to achieve a much wider dynamic range.
[0337] Running the target and endogenous control amplifications in
separate tubes and using the standard curve method requires the
least amount of optimization and validation. The advantage of using
the comparative C.sub.T method is that the need for a standard
curve is eliminated (more wells are available for samples). It also
eliminates the adverse effect of any dilution errors made in
creating the standard curve samples.
[0338] As long as the target and normalizer have similar dynamic
ranges, the comparative CT method (.DELTA..DELTA.C.sub.T method) is
the most practical method. It is expected that the normalizer will
have a higher expression level than the target (thus, a smaller
C.sub.T value). The calculations for the quantitation start with
getting the difference (.DELTA.C.sub.T) between the C.sub.T values
of the target and the normalizer:
.DELTA.C.sub.T=C.sub.T(target)-C.sub.T(normalizer)
[0339] This value is calculated for each sample to be quantitated
(unless, the target is expressed at a higher level than the
normalizer, this should be a positive value. It is no harm if it is
negative). One of these samples should be chosen as the reference
(baseline) for each comparison to be made. The comparative
.DELTA..DELTA.C.sub.T calculation involves finding the difference
between each sample's .DELTA.C.sub.T and the baseline's
.DELTA.C.sub.T. If the baseline value is representing the minimum
level of expression, the .DELTA..DELTA.C.sub.T values are expected
to be negative (because the .DELTA.C.sub.T for the baseline sample
will be the largest as it will have the greatest CT value). If the
expression is increased in some samples and decreased in others,
the .DELTA..DELTA.C.sub.T values will be a mixture of negative and
positive ones. The last step in quantitation is to transform these
values to absolute values. The formula for this is:
comparative expression level=2-.DELTA..DELTA.C.sub.T
[0340] For expressions increased compared to the baseline level
this will be something like 23=8 times increase, and for decreased
expression it will be something like 2-3=1/8 of the reference
level. Microsoft Excel can be used to do these calculations by
simply entering the CT values (there is an online ABI tutorial at
http://www.appliedbiosystems.com/support/tutorials/7700 amp/ on the
use of spread sheet programs to produce amplification plots; the
TaqMan.TM. Human Endogenous Control Plate protocol also contains
detailed instructions on using MS Excel for real-time PCR data
analysis).
[0341] The other (absolute) quantification methods are outlined in
the ABI User Bulletins
(http://docs.appliedbiosystems.com/search.taf?_UserReference=A86583271898-
50A13A.degree. C598E). The Bulletins #2 and #5 are most useful for
the general understanding of real-time PCR and quantification.
[0342] Recommendations on Procedures:
[0343] 1. Use positive-displacement pipettes to avoid inaccuracies
in pipetting,
[0344] 2. The sensitivity of real-time PCR allows detection of the
target in 2 pg of total RNA. The number of copies of total RNA used
in the reaction should ideally be enough to give a signal by 25-30
cycles (preferably less than 100 ng). The amount used should be
decreased or increased to achieve this.
[0345] 3. The optimal concentrations of the reagents are as
follows:
[0346] i. Magnesium chloride concentration should be between 4 and
7 mM. It is optimized as 5.5 mM for the primers/probes designed
using the Primer Express software.
[0347] ii. Concentrations of dNTPs should be balanced with the
exception of dUTP (if used). Substitution of dUTP for dTTP for
control of PCR product carryover requires twice dUTP that of other
dNTPs. While the optimal range for dNTPs is 500 .mu.M to 1 mM (for
one-step RT-PCR), for a typical TaqMan reaction (PCR only), 200
.mu.M of each dNTP (400 .mu.M of dUTP) is used.
[0348] iii. Typically 0.25 .mu.L (1.25 U) AmpliTaq DNA Polymerase
(5.0 U/.mu.L) is added into each 50 .mu.L reaction. This is the
minimum requirement. If necessary, optimization can be done by
increasing this amount by 0.25 U increments.
[0349] iv. The optimal probe concentration is 50-200 nM, and the
primer concentration is 100-900 nM. Ideally, each primer pair
should be optimized at three different temperatures (58, 60 and 620
C for TaqMan primers) and at each combination of three
concentrations (50, 300, 900 nM). This means setting up three
different sets (for three temperatures) with nine reactions in each
(50/50 mM, 50/300 mM, 50/900, 300/50, 300/300, 300/900, 900/50,
900/300, 900/900 mM) using a fixed amount of target template. If
necessary, a second round of optimization may improve the results.
Optimal performance is achieved by selecting the primer
concentrations that provide the lowest CT and highest .DELTA.Rn.
Similarly, the probe concentration should be optimized for 25-225
nM.
[0350] 4. If AmpliTaq Gold DNA Polymerase is being used, there has
to be a 9-12 min pre-PCR heat step at 92-950 C to activate it. If
AmpliTaq Gold DNA Polymerase is used, there is no need to set up
the reaction on ice. A typical TaqMan reaction consists of 2 min at
500 C for UNG (see below) incubation; 10 min at 95.degree. C. for
Polymerase activation, and 40 cycles of 15 sec at 95.degree. C.
(denaturation) and 1 min at 60.degree. C. (annealing and
extension). A typical reverse transcription cycle (for cDNA
synthesis), which should precede the TaqMan reaction if the
starting material is total RNA, consists of 10 min at 250 C (primer
incubation), 30 min at 48.degree. C. (reverse transcription with
conventional reverse transcriptase) and 5 min at 95.degree. C.
(reverse transcriptase inactivation).
[0351] 5. AmpErase uracil-N-glycosylase (UNG) is added in the
reaction to prevent the reamplification of carry-over PCR products
by removing any uracil incorporated into amplicons. This is why
dUTP is used rather than dTTP in PCR reaction. UNG does not
function above 55.degree. C. and does not cut single-stranded DNA
with terminal dU nucleotides. UNG-containing master mix should not
be used with one-step RT-PCR unless rTth DNA polymerase is being
used for reverse transcription and PCR (TaqMan EZ RT-PCR kit).
[0352] 6. It is necessary to include at least three No
Amplification Controls (NAC) as well as three No Template Controls
(NTC) in each reaction plate (to achieve a 99.7% confidence level
in the definition of +/-thresholds for the target amplification,
six replicates of NTCs must be run). NAC former contains sample and
no enzyme. It is necessary to rule out the presence of fluorescence
contaminants in the sample or in the heat block of the thermal
cycler (these would cause false positives). If the absolute
fluorescence of the NAC is greater than that of the NTC after PCR,
fluorescent contaminants may be present in the sample or in the
heating block of the thermal cycler.
[0353] 7. The dynamic range of a primer/probe system and its
normalizer should be examined if the .DELTA..DELTA.CT method is
going to be used for relative quantitation. This is done by running
(in triplicate) reactions of five RNA concentrations (for example,
0, 80 pg/.mu.L, 400 pg/.mu.L, 2 ng/.mu.L and 50 ng/.mu.L). The
resulting plot of log of the initial amount vs. CT values (standard
curve) should be a (near) straight line for both the target and
normalizer real-time RT-PCRs for the same range of total RNA
concentrations.
[0354] 8. The passive reference is a dye (ROX) included in the
reaction (present in the TaqMan universal PCR master mix). It does
not participate in the 5' nuclease reaction. It provides an
internal reference for background fluorescence emission. This is
used to normalize the reporter-dye signal. This normalization is
for non-PCR-related fluorescence fluctuations occurring
well-to-well (concentration or volume differences) or over time and
different from the normalization for the amount of cDNA or
efficiency of the PCR. Normalization is achieved by dividing the
emission intensity of reporter dye by the emission intensity of the
passive reference. This gives the ratio defined as Rn.
[0355] 9. If multiplexing is done, the more abundant of the targets
will use up all the ingredients of the reaction before the other
target gets a chance to amplify. To avoid this, the primer
concentrations for the more abundant target should be limited.
[0356] 10. TaqMan Universal PCR master mix should be stored at 2 to
8.degree. C. (not at -20.degree. C.).
[0357] 11. The GAPDH probe supplied with the TaqMan Gold RT-PCR kit
is labeled with a JOE reporter dye, the same probe provided within
the Pre-Developed TaqMan.TM. Assay Reagents (PDAR) kit is labeled
with VIC. Primers for these human GAPDH assays are designed not to
amplify genomic DNA.
[0358] 12. The carryover prevention enzyme, AmpErase UNG, cannot be
used with one-step RT-PCR which requires incubation at 48.degree.
C. but may be used with the EZ RT-PCR kit.
[0359] 13. One-step RT-PCR can only be used for singleplex
reactions, and the only choice for reverse transcription is the
downstream primer (not random hexamers or oligo-dT).
[0360] 14. It is ideal to run duplicates to control pipetting
errors but this inevitably increases the cost.
[0361] 15. If multiplexing, the spectral compensation option (in
Advanced Options) should be checked before the run.
[0362] 16. Normalization for the fluorescent fluctuation by using a
passive reference (ROX) in the reaction and for the amount of
cDNA/PCR efficiency by using an endogenous control (such as GAPDH,
active reference) are different processes.
[0363] 17. ABI 7700 can be used not only for quantitative RT-PCR
but also end-point PCR. The latter includes presence/absence assays
or allelic discrimination assays (such as SNP typing).
[0364] 18. Shifting Rn values during the early cycles (cycle 0-5)
of PCR means initial disequilibrium of the reaction components and
does not affect the final results as long as the lower value of
baseline range is reset.
[0365] 19. If an abnormal amplification plot has been noted
(C.sub.T value<15 cycles with amplification signal detected in
early cycles), the upper value of the baseline range should be
lowered and the samples should be diluted to increase the C.sub.T
value (a high C.sub.T value may also be due to contamination).
[0366] 20. A small .DELTA.Rn value (or greater than expected
C.sub.T value) indicates either poor PCR efficiency or low copy
number of the target.
[0367] 21. A standard deviation >0.16 for C.sub.T value
indicates inaccurate pipetting.
[0368] 22. SYBR Green entry in the Pure Dye Setup should be
abbreviated as "SYBR" in capitals. Any other abbreviation or lower
case letters will cause problems.
[0369] 23. The SDS software for ABI 7700 have conflicts with the
Macintosh Operating System version 8.1. The data should not be
analyzed on such computers.
[0370] 24. The ABI 7700 should not be deactivated for extended
periods of time. If it has ever been shutdown, it should be allowed
to warm up for at least one hour before a run. Leaving the
instrument on all times is recommended and is beneficial for the
laser. If the machine has been switched on just before a run, an
error box stating a firmware version conflict may appear. If this
happens, choose the "Auto Download" option.
[0371] 25. The ABI 7700 is only one of the real-time PCR systems
available, others include systems from BioRad, Cepheid, Corbett
Research, Roche and Stratagene.
Example 2
Determining Splice Variants
[0372] For a given gene, an anova approach to detecting splice
variants was used. The approach taken was similar to the Affymetrix
MIDAS approach. In the exon level data, there is an intensity for
each probe set, for each subject. A simple model for the intensity
would be an overall gene mean, plus a probe set effect plus a
subject effect plus error. Where i indexes the probesets and j the
subjects.
Yij=.alpha.+.beta.i+.gamma.j+.epsilon.ij
[0373] This model applies only when there is no alternate splicing.
If probe set i maps to exon e(i) and subject j is in class c(j)
then alternate splicing would be represented by the presence of a
term .delta.e(i) c(j) in the model. In X:Map annotation, probe sets
may match to multiple exons. This is associated with alternate exon
layouts in the gene, so a test for a term .delta.ic(j), that is a
probe set by class interaction, was performed. For simplicity, the
subject effect was ignored (this variation becomes part of the
noise).
Example 3
Gene Transcripts Distinguishing Sepsis from Post-Surgical
Inflammation
[0374] Any of the gene transcripts in Table 7 are able to
distinguish sepsis from post-surgical inflammation (the sign on
values in the column logFC indicates comparative up or down
regulation. By example, transcripts for ankdd1a can be expected to
be relatively up-regulated in sepsis compared to post-surgical and
transcripts for OTX1 can be expected to be relatively
down-regulated in sepsis compared to post-surgical).
Example 4
Gene Transcripts Distinguishing Sepsis from inSIRS
[0375] Any of the gene transcripts in Table 8 are able to
distinguish sepsis from inSIRS (the sign on values in the column
logFC indicates comparative up or down regulation).
Example 7
Genes Distinguishing inSIRS from Post-Surgical
[0376] Gene transcripts in Table 9 may able to distinguish inSIRS
and post-surgical inflammation (the sign on values in the column
logFC indicates comparative up or down regulation).
Example 8
Area Under Curve (AUC) for Classifiers Separating Groups Using
Exons from Splice Variants Using Several Statistical Techniques
[0377] Table 10 summarizes the area under the ROC curves (AAUC) as
percentages. The closer to 100% these are the better the
classifier.
[0378] It can be seen by looking at the percentage AUC for the
various statistical techniques that Post-surgical inflammation
versus Sepsis, Sepsis versus inSIRS, Post-surgical inflammation and
inSIRS versus Sepsis and Post-surgical versus inflammation Sepsis
and inSIRS provide good classifiers.
Example 9
Monitoring of Post-Surgical Patients
[0379] All surgery results in an acute phase response and
inflammation and the severity of the response is proportional to
the level of insult. Many cardiac surgery and abdominal surgery
patients develop a bacterial translocation and endotoxemia which
can lead to organ failure and death unless appropriately managed.
In fact, it has been demonstrated that patients with pre-existing
high plasma levels of anti-endotoxin antibody have a better
survival rate compared to those patients with low anti-endotoxin
plasma antibodies demonstrating that endotoxin and the immune
response to endotoxin play a key role in survival in these
patients. This post-surgical immune response often presents
clinically as fever. Nurses and intensivists working with
post-surgical patients with fever must therefore decide whether the
cause of the fever relates to bacterial infection. The IRC
biomarkers of the present invention, which are able to distinguish
between post-surgical inflammation, SIRS and sepsis, would
therefore be useful in determining an appropriate course of action
in such patients which could include the use of antibiotics,
anti-pyretics, immune modulators and/or anti-inflammatories.
Monitoring such patients with these biomarkers would also allow for
informed decisions on when to withdraw such treatments.
Example 10
Monitoring Trauma and Burns Patients
[0380] Severe trauma (especially head trauma) and burns patients
have high levels of tissue damage and the resultant acute phase
response and inflammation often causes swelling, fever and damage
to vital organs such as the brain and skin. Such patients are often
treated with steroids (or other anti-inflammatories) to reduce the
level of inflammation which then makes then susceptible to
bacterial infection. Brain damaged patients also often develop
fevers. A therapeutic balancing act between the use of
anti-inflammatories, immune modulating agents and antibiotics is
therefore created in these patients. The IRC biomarkers of the
present invention, which are able to distinguish between sterile
inflammation and inflammation caused by bacterial infection, are
therefore useful monitoring tools that are able to assist medical
practitioners in determining appropriate therapies for the best
outcome in such patients.
Example 11
Monitoring Patients in Intensive Care
[0381] Patients in intensive care are usually administered a number
of therapeutic compounds--many of which have opposing actions on
the immune system. Further, intensive care patients often have, or
develop, inSIRS which can lead to multi-organ failure and death.
Further still, intensive care patients often develop sepsis through
hospital acquired infection. However, the ultimate aim of intensive
care is to ensure the patient survives and is discharged to a
general ward in the minimum time. The above factors confound this
aim. Monitoring intensive care patients on a regular basis with the
IRC biomarkers of the present invention will allow medical
practitioners to: determine the level of inflammation, determine if
the patient has a hospital acquired infection, and determine
response to therapy. Information provided by these biomarkers will
therefore allow medical practitioners to tailor and modify
therapies to ensure patients survive and spend less time in
intensive care. Less time in intensive care leads to considerable
savings in medical expenses. In addition, informed use of
antibiotics leads to less usage and further savings in medical
expenses. Appropriate and informed use of antibiotics also leads to
less antibiotic resistance.
Example 12
Patients with Fever--Distinguishing Between Inflammation, inSIRS
and Sepsis
[0382] Many patients present to hospitals, or are in hospital, with
fever of unknown origin. Fever can be caused by sterile
inflammation or by microbial infection. The IRC biomarkers of the
present invention, which are able to distinguish between
inflammation, SIRS and sepsis, will be useful in screening,
stratification, diagnosing and determining appropriate therapies in
such patients.
Example 13
Determining the Severity of Immune Response to Insult
[0383] The IRC biomarkers disclosed herein are able to determine an
inflammatory response continuum from the less severe inflammatory
response of post-surgery through to the severe inflammatory
response to bacterial infection (sepsis). Determining where a
patient lies on this continuum is important with respect to
deciding what therapies (if any) should be administered.
Example 14
Provision of a Prognosis
[0384] The IRC biomarkers of the present invention permit
qualitative or quantitative grading of inflammatory response and
provide a means to separate sepsis, inSIRS and post-surgical
inflammation from each other. This, in turn, allows for the
determination of a prognosis in patients determined to have any one
of sepsis, inSIRS or post-surgical inflammation. It has been
demonstrated that in-patients with inSIRS have a 6.9 times higher
28-day mortality compared to those without SIRS (Comstedt et al.,
2007, Scand. J Trauma Resusc. Emerg. Med. 27: 17-67. 2009; Esteban
et al., 2007, Crit. Care Med. 35(5): 1284-1289). Further, with
respect to risk of dying, there is a graded severity from inSIRS to
sepsis, severe sepsis and septic shock, with an associated 28-day
mortality of approximately 10%, 20%, 20-40% and 40-60% respectively
(Brun-Buisson, C., 2000, Intensive Care Medicine 26, Suppl 1:
S64-74). Such information allows for informed decisions on choice
of therapy and how aggressively to treat.
[0385] The disclosure of every patent, patent application, and
publication cited herein is hereby incorporated herein by reference
in its entirety.
[0386] The citation of any reference herein should not be construed
as an admission that such reference is available as "Prior Art" to
the instant application.
[0387] Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
Those of skill in the art will therefore appreciate that, in light
of the instant disclosure, various modifications and changes can be
made in the particular embodiments exemplified without departing
from the scope of the present invention. All such modifications and
changes are intended to be included within the scope of the
appended claims.
TABLE-US-00002 Lengthy table referenced here
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"Sequence Listing" is available in electronic form from the USPTO
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An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
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0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
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An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References